Recombinant HCMV and RHCMV vectors and uses thereof

ABSTRACT

The present invention relates to recombinant rhesus cytomegalovirus (RhCMV) and human cytomegalovirus (HCMV) vectors encoding heterologous antigens, such as pathogen-specific antigens or tumor antigens, which may be used, for example, for the treatment or prevention of infectious disease or cancer. The recombinant RhCMV or HCMV vectors elicit and maintain high level cellular immune responses specific for the heterologous antigen while including deletions in one or more genes essential or augmenting for CMV replication, dissemination or spread.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation-in-part application of internationalpatent application PCT/US2011/036657 filed May 16, 2011 and published asWO 2011/143653 on Nov. 17, 2011, which claims priority to U.S.provisional patent application Ser. Nos. 61/334,976 filed May 14, 2010and 61/376,911 filed Aug. 25, 2010 and PCT Application PCT/US2011/029930filed Mar. 25, 2011.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appln cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

FEDERAL FUNDING LEGEND

This invention was supported, in part, by grant numbers AI088442,AI21640, AI059457, AI070890, AI070890-03S1 and AI060392 awarded by theNational Institute of Allergy and Infectious Disease of the NationalInstitutes of Health, and grant number RR00163 from the National Centerof Research Resources of the National Institutes of Health. The federalgovernment may have certain rights to this invention.

FIELD OF THE INVENTION

This invention relates to recombinant cytomegalovirus (CMV) vectors,such as human CMV (HCMV) and rhesus macaque CMV (RhCMV) vectors,encoding heterologous antigens and/or being deficient in genes that arenon-essential for growth in vivo and/or being deficient in genes thataffect replication, dissemination within the host, and spreading betweenhosts, and/or targeting to different cell types (tropism). Inparticular, the invention relates to use of recombinant CMV vectors asvaccines for the treatment or prevention of infectious disease orcancer.

BACKGROUND OF THE INVENTION

Since the emergence of the AIDS epidemic in the early 1980s,investigators have been focused on the development of an effectivevaccine for HIV. However, this effort has been wrought with difficultiesfor a variety of reasons, including 1) explosive initial HIV replicationcausing rapid systemic infection, 2) potent genetic mechanisms mediatinginnate and adaptive immune evasion, 3) genetic malleability and immuneescape, 4) host immune suppression, and 5) the ability of HIV tointegrate within the host genome and latently infect long-lived cells.These inherent characteristics of HIV have made it difficult to usetraditional vaccine approaches to generate a protective immune responseusing HIV antigens either expressed as recombinant proteins or incombination with non-replicating viral vector expression systems(prime-boost) (Barouch, D. H. 2008. Challenges in the development of anHIV-1 vaccine. Nature 455:613-619). One of the goals of this project isto generate an effective and safe HIV vaccine using HCMV as a vaccinevector.

CMV is an ubiquitous virus and a member of the beta subclass of theherpesvirus family. It is a large, double stranded DNA virus (genome ofapproximately 230 kB) that establishes life-long latent or persistentinfection. In developed countries such as the United States,approximately 70% of the population is infected by HCMV depending onsocioeconomic status. In contrast to gamma herpesviruses such asEpstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, HCMV isnon-transforming and non-oncogenic. A live, attenuated CMV vaccine(based on the human CMV Towne strain, which lacks a portion of the CMVgenome) has been administered by subcutaneous injection to over 800subjects in a phase II and III safety and efficacy trials (Arvin et al.2004 Clin. Infect. Dis. 39:233-239). While this vaccine was found to besafe, it was not completely efficacious. More recently, in an attempt toincrease its efficacy, some of the missing genes in the Towne-basedvaccine strain were replaced. This vaccine has been tested in phase IIsafety studies, and was found to be safe (Arvin et al. 2004, Clin.Infect. Dis. 39:233-239).

Although HCMV is generally benign in healthy individuals, the virus cancause devastating disease in immunocompromised populations resulting inhigh morbidity and mortality (for review, see (Pass, R. F. 2001.Cytomegalovirus, p. 2675-2705. In P. M. H. David M. Knipe, Diane E.Griffin, Robert A. Lamb Malcolm A. Martin, Bernard Roizman and StephenE. Straus (ed.), Fields Virology, 4th ed. Lippincott Williams & Wilkins,Philadelphia and Kenneson, A., and Cannon, M. J. 2007. Review andmeta-analysis of the epidemiology of congenital cytomegalovirus (CMV)infection. Rev Med Virol 17:253-276)). Recent increases in the number ofpatients undergoing immunosuppressive therapy following solid organ(SOT) or allogeneic hematopoietic cell transplantation (HCT), as well asthe expanded use of HCT for diseases such as sickle cell anemia,multiple sclerosis and solid cancers have increased the number ofpatient populations susceptible to HCMV disease (Chou, S. 1999. TransplInfect Dis 1:105-14, Nichols, W. G., and M. Boeckh. 2000. J Clin Virol16:25-40 and Sepkowitz, K. A. 2002. Clin Infect Dis 34:1098-107). HCMVis also the most common congenital viral infection, and the leadinginfectious cause of central nervous system maldevelopment in neonates(Fowler, K. B. et al. 1997. J Pediatr 130:624-30, Larke, R. P. et al.1980. J Infect Dis 142:647-53 and Peckham, C. S. et al. 1983. Lancet1:1352-5). In this regard, HCMV is considered the major cause ofsensorineural deafness in neonates independent of infectious status(Fowler, K. B. et al. 1997. J Pediatr 130:624-30). HCMV thereforeremains a major cause of mortality in multiple patient populationsemphasizing the need for new antiviral pharmacologic and vaccinestrategies. Immunity induced by natural wild-type (WT) CMV infection hasconsistently been shown unable to prevent CMV re-infection (see below).This unique characteristic of CMV presumably explains the poor efficacyof candidate vaccines in trials to prevent CMV infection (Pass, R. F. etal. 2009. N Engl J Med 360:1191-9). Nevertheless, immunity to HCMVacquired through natural infection has been shown to significantlydecrease maternal to fetal transmission of HCMV during pregnancy. Thisobservation would indicate that induction of an immunity in pregnantwomen that is comparable to that induced by natural CMV infection, butthat is induced in a safe manner, may be able to decrease maternal tofetal transmission and have a significant impact on clinical CMV diseasein the neonate. HCMV-specific T cell immunity has also been shown toafford protection against CMV disease in transplant patients, presentinganother population wherein the ability to safely induce an immunitycomparable to that acquired by natural CMV infection would have aclinical impact on CMV disease (Leen, A. M., and H. E. Heslop. 2008. BrJ Haematol 143:169-79, Riddell, S. R., and P. D. Greenberg. 2000. JAntimicrob Chemother 45 Suppl T3:35-43 and Riddell, S. R. et al. 1994.Bone Marrow Transplantation 14:78-84). Cytomegalovirus is highlyimmunogenic, but has evolved immune evasion mechanisms to enable viruspersistence and re-infection of the sero-positive host:

The immunological resources specifically devoted to controlling HCMVinfection are enormous, with CMV being one of the most immunogenicviruses known. High antibody titers are directed against the main HCMVenvelope glycoprotein (gB) during primary infection of healthyindividuals (Alberola, J. et al. 2000. J Clin Virol 16:113-22 andRasmussen, L. et al. 1991. J Infect Dis 164:835-42), and againstmultiple viral proteins (both structural and non-structural) during MCMVinfection of mice (Farrell, H. E., and G. R. Shellam. 1989. J Gen Virol70 (Pt 10):2573-86). A large proportion of the host T cell repertoire isalso directed against CMV antigens, with 5-10 fold higher median CD4⁺ Tcell response frequencies to HCMV than to acute viruses (measles, mumps,influenza, adenovirus) or even other persistent viruses such as herpessimplex and varicella-zoster viruses (Sylwester, A. W. et al. 2005. JExp Med 202:673-85). A high frequency of CD8⁺ responses to defined HCMVepitopes or proteins is also commonly observed (Gillespie, G. M. et al.2000. J Virol 74:8140-50, Kern, F. et al. 2002. J Infect Dis185:1709-16, Kern, F. et al. 1999. Eur J Immunol 29:2908-15, Kern, F. etal. 1999. J Virol 73:8179-84 and Sylwester, A. W. et al. 2005. J Exp Med202:673-85). In a large-scale human study quantifying CD4⁺ and CD8⁺ Tcell responses to the entire HCMV genome, the mean frequencies ofCMV-specific CD4⁺ and CD8⁺ T cells exceeded 10% of the memory populationfor both subsets (Sylwester, A. W. et al. 2005. J Exp Med 202:673-85).In an embodiment of the invention, it was not unusual for CMV-specific Tcells to account for >25% of the memory T cell repertoire of a specificindividual or at specific tissue sites. The clinical importance of thishigh level of CMV-specific immunity is most clearly shown by theoccurrence of multi-organ CMV disease in immune-suppressed individualsduring transplantation, and the ability of adoptive transfer of T cellsto protect these patients from CMV disease (Riddell, S. R. et al. 1994.Bone Marrow Transplantation 14:78-84).

In summary, despite the apparent safety of live, attenuated CMVvaccines, significant concerns remain with live CMV-based vaccinestrategies. Given the problems that can arise in immunosuppressedindividuals, such as AIDS patients, organ transplant recipients, orinfants who were infected in utero and that potential recipients of aCMV-based vaccine may be or become immunodeficient, significantlylimiting the utility of a live CMV vaccine. Thus, a continuing needexists for a CMV vaccine vector that is safe and efficacious in allindividuals.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

The present invention relates to recombinant vectors, advantageouslyviral vectors such as RhCMV or HCMV vectors which may comprise a nucleicacid sequence encoding a heterologous antigen, wherein the heterologousantigen is a human pathogen-specific antigen or a tumor antigen. Theantigen may be a viral antigen, a bacterial antigen, a fungal antigen, aprotozoan antigen or an antigen expressed by a hematological cancer.

The RhCMV or HCMV vectors of the present invention may containdeletions. The deletion may be in gene regions non-essential for growthin vivo. The gene regions may be in the RL11 family, the pp65 family,the US12 family and the US28 family. The deleted gene may be US2, US3,US6, US11, UL82, UL94, UL32, UL99, UL115 or UL44, or a homolog thereof.

The RhCMV vector may have deletions of Rh182-189, Rh158-166. The RhCMVvector may also have deletions in gene regions in the RL11 family, thepp65 family, the US12 family and the US28 family. In particular, theRhCMV vector may have deletions in Rh13-Rh29, Rh111-RH112, Rh191-Rh202and Rh214-Rh220, specifically Rh13.1, Rh19, Rh20, Rh23, Rh24, Rh112,Rh190, Rh192, Rh196, Rh198, Rh199, Rh200, Rh201, Rh202 and Rh220.

The RhCMV or HCMV vector may also be a tropism defective vector.Advantageously, the tropism-defective vector may lack genes required foroptimal growth in certain cell types or may contain targets fortissue-specific micro-RNAs in genes essential for viral replication. Inparticular, the tropism defective vector may have an epithelial, centralnervous system (CNS), macrophage deficient tropism or a combinationthereof.

The HCMV vector may also have deletions in gene regions in the RL11family, the pp65 family, the US12 family and the US28 family. Inparticular, the HCMV vector may have deletions in RL11, UL6, UL7, UL9,UL11, UL83 (pp65), US12, US13, US14, US17, US18, US19, US20, US21 andUL28.

Further objects of the invention include any or all of: to provideexpression products from such recombinants, methods for expressingproducts from such recombinants, compositions containing therecombinants or the expression products, methods for using theexpression products, methods for using the compositions, DNA from therecombinants, and methods for replicating DNA from the recombinants.

The present invention also relates to a method of treating a subjectwith an infectious disease, or at risk of becoming infected with aninfectious disease, or with cancer, or at risk of developing cancer,comprising selecting a subject in need of treatment and administering tothe subject the recombinant RhCMV or HCMV vector disclosed herein.

Accordingly, it is an object of the invention to not encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings.

FIG. 1 is a pair of graphs showing the percentage of CD4⁺ and CD8⁺ Tcells in CMV-positive and CMV-negative subjects that respond to HCMVORFs. Shown is the percentage of responsive T cells from totalperipheral blood (PB) T cells (top) or from memory PB T cells (bottom).

FIG. 2 is a set of graphs showing the percentage of CMV seropositivedonors with T cell responses (CD4⁺ and CD8⁺ T cells) to whole CMV orselected ORFs in PB (top); the mean response frequencies within thetotal PB T cell population to selected RhCMV ORFs (middle); and the meanresponse frequencies within the memory PB T cell population to selectedRhCMV ORFs.

FIG. 3 is a series of FACS plots showing robust peripheral blood T cellresponses to RhCMV.

FIG. 4 is a set of graphs showing peripheral blood T cell responses toRhCMV in 27 adult males. Shown are the percentage of CMV seropositive RMwith T cell responses (≧0.1% to whole CMV or selected CMV ORFs) (left);and mean T cell response frequencies to whole CMV (middle) or selectedCMV ORFs (right) among responding RM.

FIG. 5 is a series of FACS plots showing RhCMV-specific memory T cellsare highly enriched in spleen and lung. Six thousand events are shown,gated on total CD4+ or CD8+ T cells with the “%+” representing the netresponse frequencies after subtracting background within these subsets.Responding cells are all memory cells, and in the example shown, thefraction of total memory T cells (the true denominator or the response)varies from 45%/75% (CD4/CD8) in the blood to 100%/100% in lung. Thus,the response frequencies within the CD4+ and CD8+ memory subsets are asfollows: CD4 response to whole CMV(blood/spleen/lung)=0.56%/4.943%/17.4%; CD8 response to pp65A(blood/spleen/lung)=1.45%/1.69%/3.13%.

FIG. 6 is a series of graphs showing immunologic evidence of RhCMVre-infection, and depicts CMV-specific CD4⁺ and CD8⁺ T cellfrequencies/proliferative status in blood and plasma antibody titersafter re-infection.

FIG. 7 is a schematic illustration of Cre/LoxP-based recombination forconstruction of RhCMVvLoxP-based RhCMV/SIVmac239gag recombinants.

FIG. 8 is an image of an immunoblot demonstrating RhCMV/SIVgagre-infection of RhCMV-seropositive RMs.

FIG. 9 is a series of FACS plots showing rapid appearance ofgag-specific CD4+ T cells in blood after a single inoculation of CMVseropositive RM (#19997) with a first generation RhCMV(gag) vector.

FIG. 10 is a series of FACS plots showing preferential localization ofgag-specific T cells in lung after RhCMV(gag) re-infection (#21046; day56, re-infection #1).

FIG. 11 is a series of graphs showing the development of gag-specific Tcells with RhCMV(gag) re-infection (×2).

FIG. 12 is a graph showing development of anti-gag antibodies over timeafter initial re-infection.

FIG. 13 is a graph showing a comparison of gag-specific T cellfrequencies in SIV(Δnef) and RhCMV(gag)-immunized RM. In both groups,both CD4⁺ and CD8⁺ gag-specific T cell responses were higher in lung ascompared to blood, even through the percentages in blood were memorycorrected.

FIG. 14 is a series of FACS plots providing quantification andcharacterization of a young adult RM peripheral blood CD4⁺ and CD8⁺ Tcell responses to RhCMV IE-1 15mer mix by 10 color cytokine flowcytometry.

FIG. 15 is an illustration of the construction of RhCMV and HCMV vaccinevectors. Heterologous pathogen antigen(s) are inserted into RhCMV orHCMV bacterial artificial chromosomes (BACs) by E/T and Flp-mediatedrecombination.

FIG. 16 is an illustration of an exemplary tetracycline-regulatedRhCMV/HCMV safety vaccine vector. This RhCMV/HCMV safety vector containstwo interactive genetic components within the RhCMV/HCMV genome thattogether enable Tet-induced vector inactivation.

FIG. 17 is an illustration of another exemplary tetracycline-regulatedRhCMV/HCMV safety vaccine vector. Such RhCMV/HCMV vaccine vectors areconstructed by placing a gene essential for virus replication, (in thisexample, Rh70 (HCMV homologue-UL44); DNA polymerase processivityfactor), under control of the Tet-inducible system illustrated in FIG.10.

FIG. 18 is an illustration of another exemplary tetracycline-regulatedRhCMV/HCMV safety vaccine vector. Such RhCMV/HCMV vaccine vectors areconstructed containing a cytotoxic gene (CytoG) under control of theTet-inducible system as detailed in FIG. 11. After inoculation ofanimals with Tet-regulated vectors in the absence of Dox, virusreplication can be rapidly inactivated by Dox-mediated induction of thecytopathic gene resulting in death of the vaccine vector-infected cell.

FIG. 19 is an illustration of the construction of exemplary RhCMV andHCMV gene therapy vectors. Therapeutic gene(s) are inserted into RhCMVor HCMV bacterial artificial chromosomes (BACs) by E/T and Flp-mediatedrecombination. The schematic shows a generalized strategy for insertionof an epitope-tagged therapeutic gene into the non-coding region betweenrh213 and Rh214 of RhCMV. This strategy can be similarly used forinsertion of therapeutic genes at other defined sites within theRhCMV/HCMV genome.

FIG. 20 is a pair of graphs showing the T cell response to RhCMV lackingpp71. Two sero-negative RM were inoculated s.c. with 10⁷ PFU ofRhCMVΔpp71 at day 0. The CD8+ and CD4+ T cell response againstoverlapping peptides of RhCMV IE and pp65 was measured by intracellularcytokine staining in PBMC and BAL at the indicated intervals.

FIG. 21 is a graph showing miR-142-3p levels by quantitative RT-PCRanalysis from Total RNA from mouse embryo fibroblast cells (MEF), IC-21macrophage cells (IC-21), peripheral blood dendritic cells (PB DC) andbone marrow dendritic cells (BM DC). Levels of miR-142-3p were at least7000 fold higher in macrophage/dendritic cells than in MEFs. miR-142-3plevels for MEFs lie within standard background levels for ABI miRNART-PCR assays (gray section)

FIG. 22 is a graph showing the level of murine CMV (MCMV) replication ofIE3-015 control virus and IE3-142 were compared following MOI=0.1infection of 3T3 fibroblast cells. Standard plaque assays carried out on3T3 fibroblast cells.

FIG. 23 is a graph showing the level of MCMV replication of IE3-015control virus and IE3-142 were compared following MOI=0.1 infection ofIC-21 macrophage cells.

FIG. 24 is a graph showing IE1 and IE3 mRNA levels following infectionof 3T3 fibroblast with MCMV

FIG. 25 is a graph showing IE1 and IE3 mRNA levels following infectionof IC-21 macrophage cells with MCMV

FIG. 26 is a schematic showing the protocol for assessment of RhCMVshedding immunogenicity and in adult/juvenile RM.

FIG. 27 is an image of a gel showing that MCMV/VP1_(PV1) stablyexpresses VP1 in vitro.

FIG. 28 (a) is a schematic representation of MCMV/ZEBOV-NP_(CTL) andMCMV-derived vector carrying the CTL-epitope of the NP protein ofebolavirus Zaire (SEQ ID NO: 16). (b) shows a graphical representationof the results of an immunogenicity study in H2b-restricted129S1/SvlmJ/Cr mice when immunized with different MCM vector constructs.(c) shows a graphical representation of the typical responses fromMCMV/ZEBOV-NP_(CTL) vaccinated mice. The majority of ZEBOV NP-respondingT cells are polyfunctional (expressing both IFNγ and TNFα) and arespecific for the NP epitope (not observed following incubation with thePSA peptide or unstimulated controls). Consistent with MCMV infection,all mice demonstrate T cell responses to MCMV M45.

FIG. 29 a graph representing the kinetic analysis of CD8⁺ T cellresponse to MCMV/ZEBOV-NP_(CTL).

FIG. 30 is a graph showing that MCMV/ZEBOV-NP_(CTL) induces aZEBOV-specific T cell response in C57BL/6 mice.

FIG. 31 (a) is a graph showing the protective efficacy ofMCMV/ZEBOV-NP_(CTL) in terms of survival (%). (b) is a graph showing theefficacy of MCMV/ZEBOV-NP_(CTL) in terms of change in body weight (%).(c) is a graph showing the protective efficacy of MCMV/ZEBOV-NP_(CTL) interms of viremia (FFU/ml).

FIG. 32 is a gel showing a WT control and two independent clones ofMCMV/ZEBOV-NP_(CTL) (5A1 and 5D1) digested with EcoRI followed byelectrophoresis.

FIG. 33 shows the Multi-step growth analysis of MCMV/ZEBOV-NP_(CTL).

FIG. 34 shows gene regions of RhCMV that are non-essential forsuper-infection.

FIG. 35 shows immunogenicity of RhCMV/SIV vectors with deletion ofspecific genes or gene families (SIVgag- or SIVrtn-specific CD8+ T cellresponses; broncho-alveolar lavage lymphocytes).

FIG. 36 is a pair of graphs showing that the deletion of pp71 impairsviral release from normal fibroblasts, but not from fibroblastsexpressing pp71.

FIG. 37 shows the deletion of pp71 attenuates CMV-vectors in vivo butdoes not impair their ability to induce a longlasting immune responsecomparable to wildtype virus.

FIG. 38 shows that PP71-deleted CMV vectors expressing a heterologousantigen are able to super-infect CMV-infected animals and induce along-lasting immune response to the heterologous antigen but are notsecreted from infected animals.

FIG. 39 shows construction and in vitro characterization of MCMV/TetC.(a) Schematic representation of MCMV/TetC. A V5 epitope-tagged 50 kDfragment C of tetanus toxin was placed under control of the EF1αpromoter and inserted into the MCMV genome to replace the M157 genewithin the MCMV BAC, pSMfr3. (b) In vitro growth analysis ofreconstituted viruses showed replication kinetics comparable to WT MCMV.Error bars show the standard deviation (s.d.).

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand.

The instant application contains a “lengthy” Sequence Listing which hasbeen submitted via CD-R in lieu of a printed paper copy, and is herebyincorporated by reference in its entirety. Said CD-R, recorded on Jan.31, 2012, are labeled “CRF,” “Copy 1—SEQUENCE LISTING PART,” “Copy2—SEQUENCE LISTING PART,” and “Copy 3—SEQUENCE LISTING PART,”respectively, and each contains only one identical 2,727,936 byte file(43275992.txt).

In the accompanying sequence listing:

SEQ ID NO: 1 is the nucleotide sequence of RhCMV (Cercopithecineherpesvirus 8).

SEQ ID NO: 2 is the nucleotide sequence of HCMV (AD169 lab strain).

SEQ ID NO: 3 is the nucleotide sequence of HCMV (wild type strainMerlin).

SEQ ID NO: 4 is the nucleotide sequence of the Towne BAC HCMV isolate.

SEQ ID NO: 5 is the nucleotide sequence of the PH-BAC HCMV isolate.

SEQ ID NO: 6 is the nucleotide sequence of the Toledo-BAC HCMV isolate.

SEQ ID NO: 7 is the nucleotide sequence of the TR-BAC HCMV isolate.

SEQ ID NO: 8 is the nucleotide sequence of the FIX-BAC HCMV isolate.

SEQ ID NO: 9 is the nucleotide sequence of the AD 169-BAC HCMV isolate.

SEQ ID NO: 10 is the nucleotide sequence of SIV.

SEQ ID NO: 11 is the amino acid sequence of SIV gag-pol.

SEQ ID NO: 12 is the amino acid sequence of the SIV gag protein.

DETAILED DESCRIPTION OF THE INVENTION

Paradoxically, the high levels of CMV-specific immunity are unable toeither eradicate the virus from the healthy infected individual, orconfer protection of the CMV sero-positive individual againstre-infection. This ability of CMV to escape eradication by the immunesystem, and to re-infect the sero-positive host has long been believedto be linked to the multiple viral immunomodulators encoded by the virus(for review, see (Mocarski, E. S., Jr. 2002. Trends Microbiol10:332-9)). Consistent with persistent replication/chronic reactivationwithin the host, CMV also induces and maintains a characteristic andunique T cell immune response. Memory T cells induced by vaccination orinfection can be broadly characterized into either effector (T_(EM)) orcentral (T_(CM)) memory, which follow from the distinct functions ofthese two memory populations (Cheroutre, H., and L. Madakamutil. 2005.Cell Mol Life Sci 62:2853-66, Mackay, C. R. et al. 1990. J Exp Med171:801-17, Masopust, D. et al. 2001. Science 291:2413-7, Sallusto, F.et al. 1999. Nature 401:708-12 and Wherry, E. J. et al. 2003. NatImmunol 4:225-34). Embodiments of the invention that relate toimmunomodulators and the unique T-cell response elicited by HCMV arefurther described in PCT/US2011/029930.

Other embodiments of the invention relate to attenuated CMV vaccineswhich are unable or impaired in their ability to replicate in cells andtissues associated with CMV transmission and disease. The basis for thisvaccine approach is the unique ability of HCMV to induce large anddurable effector memory T cell (T_(EM)) responses to viral antigensprovides HCMV-based vectors with the potential to generate highfrequency effector site-based T cell responses that would intercept andsuppress HIV replication very early in infection, when the virus is mostvulnerable to immune control. Immunization of rhesus macaques (RMs) withreplication competent RhCMV/SIV vaccine vectors induce a large,long-lasting T_(EM) response to SIV antigens that provided protectiveimmunity to 50% of the animals following rectal challenge with highlypathogenic SIVmac239 (Hansen, S. G., Vieville, C., Whizin, N.,Coyne-Johnson, L., Siess, D. C., Drummond, D. D., Legasse, A. W.,Axthelm, M. K., Oswald, K., Trubey, C. M., et al. 2009. Effector memoryT cell responses are associated with protection of rhesus monkeys frommucosal simian immunodeficiency virus challenge. Nat Med 15:293-299)Both the anti-SIV immune response and protection mediated by theseRhCMV/SIV vectors was unprecedented compared to current vaccinecandidates, and now provides the basis for the development of aneffective HIV vaccine.

In addition, embodiments of the present invention relate to the uniqueability of RhCMV to re-infect sero+ RM in spite of the presence of asignificant anti-RhCMV immune response. In contrast, most current HIVvaccine vectors (i.e., pox and adenovirus-based vectors) are compromisedby anti-vector immunity allowing for only a single effective use ofthese vaccine platforms. This inherent property of CMV vectors can beattributed to the extensive repertoire of immune evasion genes encodedby this virus (Hansen, S. G., Powers, C. J., Richards, R., Ventura, A.B., Ford, J. C., Siess, D., Axthelm, M. K., Nelson, J. A., Jarvis, M.A., Picker, L. J., et al. 2010. Evasion of CD8+ T cells is critical forsuperinfection by cytomegalovirus. Science 328:102-106). Another furtheradvantage of CMV-based vectors is the potential to insert largecassettes expressing SIV/HIV antigens in which theoretically over 50 kbof the viral genome can be replaced with foreign DNA. Together, thesecharacteristics of CMV-based vaccine vectors have enabled development ofa RhCMV/SIV vaccine that is capable of inducing a robust T_(EM) responseto multiple SIV antigens and completely controlling viral replication inmucosally SIV-challenged RM prior to the establishment of progressive,systemic infection: a feat that has not been achieved with previousvaccine approaches.

RhCMV/SIV vector may be an effective SIV vaccine but the major concernof using a fully replication competent HCMV as an HIV vaccine vector isone of safety. Since CMV establishes a life-long infection of the host,the window for realization of any pathogenic potential of a CMV-basedvaccine extends from the time of vaccination for the life of theindividual. During this window (potentially >80 years) it is expectedthat some vaccinees will encounter periods of immune-suppression,whether this be as a consequence of iatrogenic immune conditioning priorto transplantation, or as a consequence of disease, as with HIVinfection or cancer. HCMV is also frequently shed into saliva, urine andbreast milk from healthy CMV-infected individuals for periods of timethat range from months to years. This potential of vaccine spread fromvaccinated to non-vaccinated individuals is a characteristic oflive-attenuated vaccines, such as the oral polio vaccine (OPV) (Heymann,D. L., Sutter, R. W., and Aylward, R. B. 2006. A vision of a worldwithout polio: the OPV cessation strategy. Biologicals 34:75-79). Withclear precedent from experience with OPV in the Global Polio EradicationInitiative, a potential for environmental spread will pose an additionalmajor hurdle to development of an HCMV/HIV vaccine. Thesecharacteristics of CMV must be addressed before a CMV-based vaccine willbe acceptable for general use in the human population.

Embodiments of this invention address issues of virus shedding andpathogenesis and relate to two potentially complementary approaches togenerate a safe and effective vaccines using the CMV vectors. Oneapproach focuses on development of CMV vectors that are eithercompletely or conditionally spread defective or severely restricted intheir replication, but that remain capable of inducing a protectiveimmune response against a heterologous antigen. The second approachfocuses on the generation of replication competent CMV vectors that areunable to infect epithelial cells—a cell type important for virusshedding, as well as a major cell type in the lung associated with CMVpneumonia. Some embodiments of the invention may relate to additionalsafety features into these vectors, including a block in replication inneural and myeloid cells. The optimal CMV vector will be unable to shedfrom vaccinated individuals, nor cause disease in fetuses orimmunocompromised adult RMs, but will still induce a protective immuneresponse against infectious diseases or tumors. Disclosed herein is thegeneration of an HCMV-based HIV vaccine candidate that will bepotentially highly effective against HIV infection, acceptably safe forall human target populations, non-transmissible from person to person bycontact, and therefore ready for translation into human clinical trials.

The present invention relates to HCMV and RhCMV recombinant vectors thatencode heterologous antigens that elicit and maintain high levelcellular and humoral immune responses specific for the encoded antigen.The present invention also relates to attenuated CMV vaccines which arelimited in their ability to replicate in cells and tissues associatedwith CMV transmission and disease.

Further objects of the invention include any or all of: providingexpression products from such recombinants, methods for expressingproducts from such recombinants, compositions containing therecombinants or the expression products, methods for using theexpression products, methods for using the compositions, DNA from therecombinants, and methods for replicating DNA from the recombinants.

Thus, provided herein are recombinant RhCMV or HCMV vectors including anucleic acid sequences encoding heterologous antigens. In someembodiments, the heterologous antigen is a pathogen-specific antigen. Inother embodiments, the heterologous antigen is a tumor antigen. In someembodiments, the disclosed vectors include a deletion in one or more CMVgenes encoding an immunomodulatory protein. In some embodiments, thedisclosed CMV vectors include a deletion in one or more genes essentialfor CMV replication. Further provided are compositions comprising therecombinant RhCMV or HCMV vectors and a pharmaceutically acceptablecarrier as well as the use of such composition in treating a subject.

Also provided is a method of treating a subject with an infectiousdisease, or at risk of becoming infected with an infectious disease, anda method of treating a subject with cancer, or at risk of developingcancer. The methods include selecting a subject in need of treatment andadministering to the subject a recombinant RhCMV or HCMV vector encodinga heterologous antigen, or a composition thereof.

Further provided are recombinant RhCMV or HCMV vectors including adeletion in one or more RhCMV or HCMV genes that are essential for oraugments replication. In some embodiments, at least one essential oraugmenting gene is UL82, UL94, UL32, UL99, UL115 or UL44, or a homologthereof. In some embodiments, the recombinant RhCMV or HCMV vectorsfurther include a heterologous antigen, such as a pathogen-specificantigen or a tumor antigen. Recombinant RhCMV and HCMV vectors having adeletion in an essential gene and encoding a heterologous antigen can beused, for example, as vaccines for the treatment of infectious diseaseor cancer. In the absence of a heterologous antigen, the recombinantRhCMV and HCMV vectors having a deletion in an essential gene can beused, for example, for vaccination against CMV.

Further provided are attenuated virus vaccines that are unable toreplicate in cells and tissues associated with CMV transmission anddisease. Live RhCMV/SIV vectors with a near wildtype genetic backgroundprovide an effective vaccine that induces SIV protective immunity inrhesus macaques. For a human CMV (HCMV)/HIV vaccine to be safe for allpotential subjects in a general population, including individuals withunsuspected immune compromise, the CMV vaccine vector needs to beattenuated without losing the ability to induce protective immunity. CMVcan replicate in a wide variety of cells and tissues in the host,including: neurons in the central nervous system (CNS), epithelialcells, hepatocytes, lung and kidney. Myeloid and endothelial cells areconsidered persistent sites for CMV in the host. During overt CMVdisease in immunocompromised individuals, direct infection resulting indestruction of epithelial and endothelial cells in the lung, liver andretina is responsible for disease in these target organs. Duringcongenital infection, direct CMV infection of neuronal cells is believedto account for the associated hearing deficits and mental retardation.Embodiments of the invention relate to modulating the ability of CMV toreplicate in these critical cell types in order to increase vectorsafety without compromising vaccine efficacy, said attenuated virusesand their use as vaccines.

Embodiments of the invention relate to HCMV as a vector for inducingprotective immunity to HIV, which is based on the highly innovativehypothesis that a high frequency, effector memory-biased T cell responsehas distinct advantages over conventional vaccine generated memory inprotecting against lentiviral infections, combined with the recognitionthat HCMV provides just such a response. This characteristic of HCMV isunique to this virus, even when compared to other persistent virusessuch as herpes simplex virus (HSV) and Epstein-Barr virus (Asanuma, H.,Sharp, M., Maecker, H. T., Maino, V. C., and Arvin, A. M. 2000.Frequencies of memory T cells specific for varicella-zoster virus,herpes simplex virus, and cytomegalovirus by intracellular detection ofcytokine expression. J Infect Dis 181:859-866; Harari, A., Vallelian,F., Meylan, P. R., and Pantaleo, G. 2005. Functional heterogeneity ofmemory CD4 T cell responses in different conditions of antigen exposureand persistence. J Immunol 174:1037-1045; Harari, A., Enders, F. B.,Cellerai, C., Bart, P. A., and Pantaleo, G. 2009. Distinct profiles ofcytotoxic granules in memory CD8 T cells correlate with function,differentiation stage, and antigen exposure. J Virol 83:2862-2871;Sylwester, A. W., Mitchell, B. L., Edgar, J. B., Taormina, C., Pelte,C., Ruchti, F., Sleath, P. R., Grabstein, K. H., Hosken, N. A., Kern,F., et al. 2005. Broadly targeted human cytomegalovirus-specific CD4+and CD8+ T cells dominate the memory compartments of exposed subjects. JExp Med 202:673-685.).

Even though CMV is benign in immunocompetent individuals, in order toextend these findings into an HCMV/HIV vaccine for human testing, wecertain aspects of the CMV vector need to attenuate. Classically,attenuated viral vaccines have been generated through serial passagingof viruses through cells in culture. This approach is tedious andevidence with oral polio vaccine (OPV) emphasizes the safety concernsregarding reversion of virus vaccines attenuated by such ‘blind’ passageinto a pathogenic phenotype (Rahimi, P., Tabatabaie, H., Gouya, M. M.,Zahraie, M., Mahmudi, M., Ziaie, A., Rad, K. S., Shahmahmudi, S.,Musavi, T., Azad, T. M., et al. 2007. Characterization of mutations inthe VP(1) region of Sabin strain type 1 polioviruses isolated fromvaccine-associated paralytic poliomyelitis cases in Iran. J Clin Virol39:304-307; Kew, O., Morris-Glasgow, V., Landaverde, M., Burns, C.,Shaw, J., Garib, Z., Andre, J., Blackman, E., Freeman, C. J., Jorba, J.,et al. 2002. Outbreak of poliomyelitis in Hispaniola associated withcirculating type 1 vaccine-derived poliovirus. Science 296:356-359.).

Early live attenuated vaccines to HCMV were generated over 30 years agothrough serial passage of virus in tissue culture. These HCMV vaccineswere tested in human volunteers and transplant patients (Quinnan, G. V.,Jr., Delery, M., Rook, A. H., Frederick, W. R., Epstein, J. S.,Manischewitz, J. F., Jackson, L., Ramsey, K. M., Mittal, K., Plotkin, S.A., et al. 1984. Comparative virulence and immunogenicity of the Townestrain and a nonattenuated strain of cytomegalovirus. Ann Intern Med101:478-483; Plotkin, S. A., Smiley, M. L., Friedman, H. M., Starr, S.E., Fleisher, G. R., Wlodaver, C., Dafoe, D. C., Friedman, A. D.,Grossman, R. A., and Barker, C. F. 1984. Towne-vaccine-inducedprevention of cytomegalovirus disease after renal transplants. Lancet1:528-530; Plotkin, S. A., Starr, S. E., Friedman, H. M., Gonczol, E.,and Weibel, R. E. 1989. Protective effects of Towne cytomegalovirusvaccine against low-passage cytomegalovirus administered as a challenge.J Infect Dis 159:860-865).

While the HCMV vaccine may be considered safe, concerns still remainregarding both pathogenicity as well as the ability of the virus tospread to unvaccinated sero-negative individuals. The ability torationally design an HCMV vaccine that is less pathogenic and not shedinto the environment is now available with the advent of technologicalbreakthroughs to clone and genetically manipulate CMV. With a long-termgoal of generating a CMV vaccine vector encoding HIV antigens that issafe and unable to spread to other individuals. Embodiments of thisinvention relate to the rational design and use of the latest bacterialgenetic techniques to generate a CMV-based vector that has a restrictedtropism for cells involved in shedding as well as an altered ability toreplicate in tissues associated with both adult and congenital CMVdisease.

One embodiment of the invention relates to alteration of thecell-tropism of the CMV vector so as to prevent infection of specificcell types involved in potential tissue damage and/or shedding intourine or secretions. CMV is capable of infecting a wide variety of cellsin the host, including: epithelial cells in gut, kidney, lung andretina, neuronal cells in the CNS, hepatocytes, as well as endothelialcells and myeloid lineage cells that are considered persistent sites ofthe virus (Dankner, W. M., McCutchan, J. A., Richman, D. D., Hirata, K.,and Spector, S. A. 1990. Localization of human cytomegalovirus inperipheral blood leukocytes by in situ hybridization. J Infect Dis161:31-36; Einhorn, L., and Ost, A. 1984. Cytomegalovirus infection ofhuman blood cells. J Infect Dis 149:207-214; Gnann, J. W., Jr., Ahlmen,J., Svalander, C., Olding, L., Oldstone, M. B., and Nelson, J. A. 1988.Inflammatory cells in transplanted kidneys are infected by humancytomegalovirus. Am J Pathol 132:239-248; Howell, C. L., Miller, M. J.,and Martin, W. J. 1979. Comparison of rates of virus isolation fromleukocyte populations separated from blood by conventional andFicoll-Paque/Macrodex methods. J Clin Microbiol 10:533-537; Myerson, D.,Hackman, R. C., Nelson, J. A., Ward, D. C., and McDougall, J. K. 1984.Widespread presence of histologically occult cytomegalovirus. Hum Pathol15:430-439; Schrier, R. D., Nelson, J. A., and Oldstone, M. B. 1985.Detection of human cytomegalovirus in peripheral blood lymphocytes in anatural infection. Science 230:1048-1051; Sinzger, C., Grefte, A.,Plachter, B., Gouw, A. S., The, T. H., and Jahn, G. 1995. Fibroblasts,epithelial cells, endothelial cells and smooth muscle cells are majortargets of human cytomegalovirus infection in lung and gastrointestinaltissues. J Gen Virol 76:741-750.).

HCMV encodes >200 genes and several of the genes that are dispensablefor basic virus replication have been identified as tropism determinantsthat enable the virus to enter and replicate in macrophages, endothelialcells, and epithelial cells. One locus of HCMV genes, UL128-131A, hasbeen shown to be essential for entry into endothelial and epithelialcells (Gerna, G., Percivalle, E., Lilleri, D., Lozza, L., Fornara, C.,Hahn, G., Baldanti, F., and Revello, M. G. 2005. Dendritic-cellinfection by human cytomegalovirus is restricted to strains carryingfunctional UL131-128 genes and mediates efficient viral antigenpresentation to CD8+ T cells. J Gen Virol 86:275-284; Hahn, G., Revello,M. G., Patrone, M., Percivalle, E., Campanini, G., Sarasini, A., Wagner,M., Gallina, A., Milanesi, G., Koszinowski, U., et al. 2004. Humancytomegalovirus UL131-128 genes are indispensable for virus growth inendothelial cells and virus transfer to leukocytes. J Virol78:10023-10033; Wang, D., and Shenk, T. 2005. Human cytomegalovirusUL131 open reading frame is required for epithelial cell tropism. JVirol 79:10330-10338; Wang, D., and Shenk, T. 2005. Humancytomegalovirus virion protein complex required for epithelial andendothelial cell tropism. Proc Natl Acad Sci USA 102:18153-18158;Ryckman, B. J., Rainish, B. L., Chase, M. C., Borton, J. A., Nelson, J.A., Jarvis, M. A., and Johnson, D. C. 2008. Characterization of thehuman cytomegalovirus gH/gL/UL128-131 complex that mediates entry intoepithelial and endothelial cells. J Virol 82:60-70; Ryckman, B. J.,Jarvis, M. A., Drummond, D. D., Nelson, J. A., and Johnson, D. C. 2006.Human cytomegalovirus entry into epithelial and endothelial cellsdepends on genes UL128 to UL150 and occurs by endocytosis and low-pHfusion. J Virol 80:710-722.).

The RhCMV homologues for HCMV UL128 and 130 are inactivated in the RhCMVstrain 68-1 used as the backbone vector for our RhCMV/SIV studies(Lilja, A. E., and Shenk, T. 2008. Efficient replication of rhesuscytomegalovirus variants in multiple rhesus and human cell types. ProcNatl Acad Sci USA 105:19950-19955). Interestingly, RhCMV 68-1 stillgrows in epithelial and endothelial cells (albeit at a reduced ratecompared to low passage RhCMV virus with intact UL128/130) (Lilja, A.E., Chang, W. L., Barry, P. A., Becerra, S. P., and Shenk, T. E. 2008.Functional genetic analysis of rhesus cytomegalovirus: Rh01 is anepithelial cell tropism factor. J Virol 82:2170-2181; Rue, C. A.,Jarvis, M. A., Knoche, A. J., Meyers, H. L., DeFilippis, V. R., Hansen,S. G., Wagner, M., Fruh, K., Anders, D. G., Wong, S. W., et al. 2004. Acyclooxygenase-2 homologue encoded by rhesus cytomegalovirus is adeterminant for endothelial cell tropism. Journal of Virology78:12529-12536.), but does show reduced shedding compared to low passageRhCMV suggesting that reducing epithelial/endothelial cell tropism mayattenuate the virus. Mutational analysis of RhCMV 68-1 has identified 4other RhCMV genes [Rh01 (HCMV TLR1), Rh159 (HCMV UL148), Rh160 (UL132)and Rh203 (HCMVUS22)] that are also required for epithelial celltropism. Embodiments of the invention relate to the mutation of theremainder of these epithelial cell tropism genes to highly reduce, ifnot abrogate, the ability of CMV to infect epithelial cells, therebypreventing its ability to be shed into urine or glandular secretions(i.e., saliva and breast milk), yet likely not compromise the ability ofa CMV vector to induce a protective immune response to HIV/SIV.

Moreover, since CMV infection of epithelial cells in the lung and retinaresults in pneumonia and retinitis, respectively, elimination of all theCMV epithelial cell tropism genes may significantly reduce the resultantvector's pathogenic potential. Aspects of the invention relate to thishighly targeted and innovative approach that will significantly enhanceboth the safety of the RhCMV/HCMV vector for use as an SIV/HIV vaccine,as well as prevent shedding and the potential spread of the vaccinevector into the unvaccinated population.

Further embodiments relate to exploiting the tissue-specific expressionof cellular microRNAs (miRNAs) to attenuate the virus in tissuesassociated with disease in adult and congenital infection. miRNAs aresmall noncoding 21-22 bp RNAs that are highly conserved and expressed inall animal cells from drosophila to humans and therefore RM (Bartel, D.P. 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell116:281-297). miRNAs are an ancient system for posttranscriptionalregulation that are involved in a wide range of biological processes andregulate gene expression by binding target sequences in the 3′ UTR ofmRNAs causing either inhibition of translation of destabilization of themRNA (Bartel, D. P. 2009. MicroRNAs: target recognition and regulatoryfunctions. Cell 136:215-233). These RNA species are also encoded in DNAviruses such as CMV and expression and function of these miRNAs ischaracterized as described in Dunn, W., Trang, P., Zhong, Q., Yang, E.,van Belle, C., and Liu, F. 2005. Human cytomegalovirus expresses novelmicroRNAs during productive viral infection. Cell Microbiol 7:1684-1695;Grey, F., Antoniewicz, A., Allen, E., Saugstad, J., McShea, A.,Carrington, J. C., and Nelson, J. 2005. Identification andcharacterization of human cytomegalovirus-encoded microRNAs. J Virol79:12095-12099; Grey, F., Meyers, H., White, E. A., Spector, D. H., andNelson, J. 2007. A human cytomegalovirus-encoded microRNA regulatesexpression of multiple viral genes involved in replication. PLoS Pathog3:e163 and Pfeffer, S., Sewer, A., Lagos-Quintana, M., Sheridan, R.,Sander, C., Grasser, F. A., van Dyk, L. F., Ho, C. K., Shuman, S.,Chien, M., et al. 2005. Identification of microRNAs of the herpesvirusfamily. Nat Methods 2:269-276.

Mammalian miRNAs can either be expressed ubiquitously in all tissues ofthe host, expressed only during certain times during embryogenesis inwhich these miRNA species play a major role in developmental processes,or can be expressed only in a tissue-specific manner (such as miR-142-3pin myeloid lineage cells, miR-124 in CNS tissue, and miR-122 in liver)(Brown, B. D., Gentner, B., Cantore, A., Colleoni, S., Amendola, M.,Zingale, A., Baccarini, A., Lazzari, G., Galli, C., and Naldini, L.2007. Endogenous microRNA can be broadly exploited to regulate transgeneexpression according to tissue, lineage and differentiation state. NatBiotechnol 25:1457-1467; Barnes, D., Kunitomi, M., Vignuzzi, M.,Saksela, K., and Andino, R. 2008. Harnessing endogenous miRNAs tocontrol virus tissue tropism as a strategy for developing attenuatedvirus vaccines. Cell Host Microbe 4:239-248; Lee, C. Y., Rennie, P. S.,and Jia, W. W. 2009. MicroRNA regulation of oncolytic herpes simplexvirus-1 for selective killing of prostate cancer cells. Clin Cancer Res15:5126-5135; Perez, J. T., Pham, A. M., Lorini, M. H., Chua, M. A.,Steel, J., and tenOever, B. R. 2009. MicroRNA-mediated species-specificattenuation of influenza A virus. Nat Biotechnol 27:572-576.).

Tissue specific expression of miRNAs is exploited to generate anattenuated polio vaccine through the introduction of multiple miRNAtarget sequences of miR-124 that is specifically expressed in the CNSinto the 3′UTR of the poliovirus genome (Barnes, D., Kunitomi, M.,Vignuzzi, M., Saksela, K., and Andino, R. 2008. Harnessing endogenousmiRNAs to control virus tissue tropism as a strategy for developingattenuated virus vaccines. Cell Host Microbe 4:239-248). Addition of themiR-124 target sequences to the poliovirus genome was observed tosignificantly attenuate virus infection in mice. Similarly, multipletarget sequences of miR-93 that is ubiquitously expressed in allmammalian but not avian tissues were added to the nucleoprotein gene ofinfluenza resulting in a species-restricted influenza mutant that wasable to grow in chicken eggs but not in mice (Perez, J. T., Pham, A. M.,Lorini, M. H., Chua, M. A., Steel, J., and tenOever, B. R. 2009.MicroRNA-mediated species-specific attenuation of influenza A virus. NatBiotechnol 27:572-576).

Embodiments of this invention relate to this attenuation approach beingeffective for larger viruses, such as murine CMV (MCMV). Unlike thesmall RNA viruses, CMV encodes over 200 genes of which approximately 50%are essential and necessary for replication or encode structuralproteins of the virus. One of these essential MCMV genes is theimmediate early (IE) 3 gene (the mouse correlate of IE2 in HCMV orRhCMV) that encodes a transcriptional regulatory protein necessary forsubsequent activation of early and late genes in the virus. Deletion ofthis gene completely blocks viral replication in cells and mouse tissues(Angulo, A., Ghazal, P., and Messerle, M. 2000. The majorimmediate-early gene ie3 of mouse cytomegalovirus is essential for viralgrowth. J Virol 74:11129-11136;). It is described herein thatintroduction of target sequences of tissue-specific miRNAs into the3′UTR of this gene would attenuate viral replication in these cells.

A further embodiment relates to target sequences of miR-142-3p beingexpressed only in myeloid lineage cells (Brown, B. D., Gentner, B.,Cantore, A., Colleoni, S., Amendola, M., Zingale, A., Baccarini, A.,Lazzari, G., Galli, C., and Naldini, L. 2007. Endogenous microRNA can bebroadly exploited to regulate transgene expression according to tissue,lineage and differentiation state. Nat Biotechnol 25:1457-1467). Myeloidlineage cells have been shown to represent a reservoir of latent virus,and are thought to harbor and disseminate virus throughout the host(Jarvis, M. A., and Nelson, J. A. 2002. Mechanisms of humancytomegalovirus persistence and latency. Front Biosci 7:d1575-1582).Further studies with MCMV (Snyder, C. M., Allan, J. E., Bonnett, E. L.,Doom, C. M., and Hill, A. B. Cross-presentation of a spread-defectiveMCMV is sufficient to prime the majority of virus-specific CD8+ T cells.PLoS One 5:e9681) indicate that cross-priming is the primary mechanismby which CMV-encoded proteins prime the immune response, replication inmyeloid dendritic cells may have a surprisingly minimal impact on CMVimmunogenicity.

Bacterial artificial chromosome (BAC)-based technology is used togenerate a recombinant MCMV virus that contained four repeated targetsequences (four 21mers) with exact complementarity to the cellularmiRNA, miR-142-3p, within the 3′UTR of the essential viral gene IE3(IE3-142). To confirm the extent to which miR-142-3p expression couldrepress IE3-142 replication, virus growth assays are performed in themacrophage cell line, IC-21. RT-PCR analysis confirmed that IC-21 cellsexpress high levels of miR-142-3p (FIG. 21) making the cell linesuitable to test the effectiveness of the strategy. Preliminaryexperiments confirmed the utility of the approach for cell-type specificattenuation of CMV. Although IE3-142 replicated to wild type levels infibroblasts, growth was completely blocked in IC-21 macrophage cells(FIGS. 22 and 23). A control virus, IE3-015, which contains only vectorsequence within the IE3 insertion site, replicates to wild-type levelsin IC-21 cells. RT-PCR analysis indicates that IE3 expression wascompletely abrogated following infection of IC-21 cells, but notfollowing infection of fibroblast cells (lacking miR-142-3p expression)indicating that disruption of IE3 expression is not due to insertion ofthe target sequence.

Embodiments of the invention relate to strategy to attenuate CMV basedon the showing that viruses can be attenuated for tissue-specific growthby using miRNA target sequences and the attenuation of MCMV in myeloidcells through the targeting of cell specific miRNAs to essential viralgenes. Since the CNS is a major target for CMV pathogenesis in bothcongenital and adult disease, RhCMV/SIV vaccines and HCMV/HIV aregenerated that contain target sequences of highly conserved miRNAsspecifically expressed by neurons fused to essential CMV genes toprevent replication in the CNS. Target sequences of the myeloid miRNAmiR-124 to prevent replication and dissemination of the CMV vector inthis cell type are also used. Together, these attenuated viruses willprovide a further level of safety that will enable the use of thisvaccine in all human target populations.

Besides the translational application of using miRNA tissue-specificexpression to generate safe CMV vectors, the tools are available for thefirst time to ask important scientific questions, most notably includingthe determination of which cell types are required for establishment andpersistence of CMV infection, induction of T cell immunity, and diseaseas described herein. Embodiments of the invention relate to theinfection of particular cell types that may be crucial for generation ofthe high frequency T_(EM)-biased T cell immunity characteristic of CMVelicited responses, and determination of how restriction of viraltropism changes the character of CMV disease.

Some non-limiting abbreviations include:

BAC Bacterial artificial chromosome

BAL Bronchioalveolar lavage

CM Central memory

CMV Cytomegalovirus

CytoG Cytotoxic gene

Dox Doxycycline

ELISA Enzyme linked immunosorbent assay

EM Effector memory

HCMV Human cytomegalovirus

HCV Hepatitis C virus

HIV Human immunodeficiency virus

ICS Intracellular cytokine staining

i.p. Intraperitoneal

MCMV Murine cytomegalovirus

MEF Murine embryonic fibroblast

MOI Multiplicity of infection

NHP Non-human primate

NP Nucleoprotein

NS Non-structural

ORF Open reading frame

PB Peripheral blood

PBMC Peripheral blood mononuclear cell

PCR Polymerase chain reaction

PFU Plaque forming unit

RhCMV Rhesus cytomegalovirus

RM Rhesus macaque

s.c. Subcutaneous

SIV Simian immunodeficiency virus

Tet Tetracycline

WT Wild-type

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of theinvention, the following terminology may be used:

Adjuvant: A substance or vehicle that non-specifically enhances theimmune response to an antigen or other composition. Adjuvants caninclude a suspension of minerals (alum, aluminum hydroxide, orphosphate) on which antigen is adsorbed; or water-in-oil emulsion inwhich antigen solution is emulsified in mineral oil (for example,Freund's incomplete adjuvant), sometimes with the inclusion of killedmycobacteria (Freund's complete adjuvant) to further enhanceantigenicity. Immunostimulatory oligonucleotides (such as thoseincluding a CpG motif) can also be used as adjuvants (for example, seeU.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116;6,339,068; 6,406,705; and 6,429,199). Adjuvants also include biologicalmolecules, such as costimulatory molecules. Exemplary biologicaladjuvants include IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3,CD72, B7-1, B7-2, OX-40L and 41 BBL.

Administration: The introduction of a composition into a subject by achosen route. For example, if the chosen route is intravenous, thecomposition is administered by introducing the composition into a veinof the subject. Administration can be local or systemic. Examples oflocal administration include, but are not limited to, topicaladministration, subcutaneous administration, intramuscularadministration, intrathecal administration, intrapericardialadministration, intra-ocular administration, topical ophthalmicadministration, or administration to the nasal mucosa or lungs byinhalational administration. Systemic administration includes any routeof administration designed to distribute an active compound orcomposition widely throughout the body via the circulatory system. Thus,systemic administration includes, but is not limited to intraperitoneal,intra-arterial and intravenous administration. Systemic administrationalso includes, but is not limited to, topical administration,subcutaneous administration, intramuscular administration, oradministration by inhalation, when such administration is directed atabsorption and distribution throughout the body by the circulatorysystem.

Animal: A living multi-cellular vertebrate or invertebrate organism, acategory that includes, for example, mammals and birds. The term mammalincludes both human and non-human mammals. The term “primate” includesboth human and non-human primates. “Non-human primates” are simianprimates such as monkeys, chimpanzees, orangutans, baboons, andmacaques. Similarly, the term “subject” includes both human andveterinary subjects, such as non-human primates.

Antibody: Antibodies includes immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, forinstance, molecules that contain an antigen binding site thatspecifically binds (immunoreacts with) an antigen. A naturally occurringantibody (for example, IgG, IgM, IgD) includes four polypeptide chains,two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds. However, it has been shown that the antigen-bindingfunction of an antibody can be performed by fragments of a naturallyoccurring antibody. Thus, these antigen-binding fragments are alsointended to be designated by the term “antibody.” Specific, non-limitingexamples of binding fragments encompassed within the term antibodyinclude (i) an Fab fragment consisting of the VL, VH, CL, and CH1domains; (ii) an Fd fragment consisting of the VH and CH1 domains; (iii)an Fv fragment consisting of the VL and VH domains of a single arm of anantibody, (iv) a dAb fragment (Ward et al., Nature 341:544-546, 1989)which consists of a VH domain; (v) an isolated complementaritydetermining region (CDR); and (vi) an F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. The term “antigen”includes all related antigenic epitopes. “Epitope” or “antigenicdeterminant” refers to a site on an antigen to which B and/or T cellsrespond. In one embodiment, T cells respond to the epitope, when theepitope is presented in conjunction with an MHC molecule. Epitopes canbe formed both from contiguous amino acids or noncontiguous amino acidsjuxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained on exposure to denaturingsolvents whereas epitopes formed by tertiary folding are typically loston treatment with denaturing solvents. An epitope typically includes atleast 3, and more usually, at least 5, about 9, or about 8-10 aminoacids in a unique spatial conformation. Methods of determining spatialconformation of epitopes include, for example, x-ray crystallography and2-dimensional nuclear magnetic resonance.

In some embodiments, an antigen is a polypeptide specifically expressedin tumor cells (i.e., a tumor antigen). In some cases, tumor antigensare also expressed in normal cells, but the expression level in normalcells is significantly lower than the expression level in tumor cells.In some embodiments, the antigen is a pathogen-specific antigen. In thecontext of the present disclosure, a pathogen-specific antigen is anantigen that elicits an immune response against the pathogen and/or isunique to a pathogen (such as a virus, bacterium, fungus or protozoan).

Antigenic fragment: Refers to any portion of a protein of polypeptidethat is capable of eliciting an immune response.

Antigen-specific T cell: A CD8+ or CD4+ lymphocyte that recognizes aparticular antigen. Generally, antigen-specific T cells specificallybind to a particular antigen presented by MHC molecules, but not otherantigens presented by the same MHC.

Attenuated: In the context of a live virus, the virus is attenuated ifits ability to infect a cell or subject and/or its ability to producedisease is reduced (for example, eliminated) compared to a wild-typevirus. Typically, an attenuated virus retains at least some capacity toelicit an immune response following administration to an immunocompetentsubject. In some cases, an attenuated virus is capable of eliciting aprotective immune response without causing any signs or symptoms ofinfection. In some embodiments, the ability of an attenuated virus tocause disease in a subject is reduced at least about 10%, at least about25%, at least about 50%, at least about 75% or at least about 90%relative to wild-type virus.

Cancer, tumor, neoplasia and malignancy: A neoplasm is an abnormalgrowth of tissue or cells that results from excessive cell division.Neoplastic growth can produce a tumor. The amount of a tumor in anindividual is the “tumor burden” which can be measured as the number,volume, or weight of the tumor. A tumor that does not metastasize isreferred to as “benign.” A tumor that invades the surrounding tissueand/or can metastasize is referred to as “malignant.” Malignant tumorsare also referred to as “cancer.”

Hematologic cancers are cancers of the blood or bone marrow. Examples ofhematological (or hematogenous) cancers include leukemias, includingacute leukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia. In some cases, lymphomas are considered solid tumors.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and othersarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreaticcancer, breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma,papillary thyroid carcinoma, pheochromocytomas sebaceous glandcarcinoma, papillary carcinoma, human papilloma virus (HPV)-infectedneoplasias, papillary adenocarcinomas, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor,seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma(such as brainstem glioma and mixed gliomas), glioblastoma (also knownas glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma,medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,neuroblastoma, retinoblastoma and brain metastasis).

CMV (cytomegalovirus): A member of the beta subclass of the family ofherpesviruses. CMV is a large (˜230 kB genome), double stranded DNAvirus, with host-range specific variants such as MCMV (murine CMV),RhCMV (rhesus CMV) and HCMV (human CMV). In the context of the presentinvention, “RhCMV” refers to any strain, isolate or variant of rhesusCMV. In particular examples, RhCMV comprises the nucleotide sequence ofSEQ ID NO: 1, or a sequence that is at least 80%, at least 85%, at least90%, at least 95%, or at least 99% identical to SEQ ID NO: 1. As usedherein, “HCMV” includes any strain, isolate or variant of human CMV. Inparticular examples, HCMV comprises the nucleotide sequences of any oneof SEQ ID NOs: 2-9, or a sequence that is at least 80%, at least 85%, atleast 90%, at least 95%, or at least 95% identical to any one of SEQ IDNOs: 2-9.

Chemotherapy: In cancer treatment, chemotherapy refers to theadministration of one or more agents to kill or slow the reproduction ofrapidly multiplying cells, such as tumor or cancer cells. In aparticular example, chemotherapy refers to the administration of one ormore anti-neoplastic agents to significantly reduce the number of tumorcells in the subject, such as by at least 50%.

Chemotherapeutic agent: An agent with therapeutic usefulness in thetreatment of diseases characterized by abnormal cell growth orhyperplasia. Such diseases include cancer, autoimmune disease as well asdiseases characterized by hyperplastic growth such as psoriasis. One ofskill in the art can readily identify a chemotherapeutic agent (forinstance, see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86in Harrison's Principles of Internal Medicine, 14th edition; Perry etal., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nd ed., ©2000Churchill Livingstone, Inc; Baltzer L, Berkery R (eds): Oncology PocketGuide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; FischerD S, Knobf M F, Durivage H J (eds): The Cancer Chemotherapy Handbook,4th ed. St. Louis, Mosby-Year Book, 1993).

Decrease or deplete: To reduce the quality, amount, or strength ofsomething. In one example, a therapy (such as the methods providedherein) decreases a tumor (such as the size of a tumor, the number oftumors, the metastasis of a tumor, the reoccurrence of a tumor orcombinations thereof), or one or more symptoms associated with a tumor,for example as compared to the response in the absence of the therapy.In a particular example, a therapy decreases the size of a tumor, thenumber of tumors, the metastasis of a tumor, the reoccurrence of a tumoror combinations thereof, subsequent to the therapy, such as a decreaseof at least 10%, at least 20%, at least 50%, or even at least 90%.Similarly, in other embodiments, a therapy decreases the infectious loador titer of a pathogen, or one or more symptoms associated withinfection.

Deletion: The removal of a sequence of DNA, the regions on either sideof the removed sequence being joined together.

Effective amount: A quantity sufficient to achieve a desired effect in asubject being treated. An effective amount of a composition, such as avaccine, can be administered in a single dose, or in several doses,during a course of treatment. However, the effective amount of thecompound will be dependent on the compound applied, the subject beingtreated, the severity and type of the affliction, and the manner ofadministration of the compound.

Epitope: An epitope of interest is an antigen or immunogen orimmunologically active fragment thereof from a pathogen or toxin ofveterinary or human interest. An epitope of interest can be an antigenof pathogen or toxin, or from an antigen of a pathogen or toxin, oranother antigen or toxin which elicits a response with respect to thepathogen, of from another antigen or toxin which elicits a response withrespect to the pathogen.

Expression: Translation of a nucleic acid into a protein, for examplethe translation of a mRNA encoding a tumor-specific or pathogen-specificantigen into a protein.

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked, for example the expression of a heterologouspolynucleotide spliced in a CMV genome and encoding an antigenic proteinoperably linked to expression control sequences. Expression controlsequences are operatively linked to a nucleic acid sequence when theexpression control sequences control and regulate the transcription and,as appropriate, translation of the nucleic acid sequence. Thusexpression control sequences can include appropriate promoters,enhancers, transcription terminators, a start codon (ATG) in front of aprotein-encoding gene, splicing signal for introns, maintenance of thecorrect reading frame of that gene to permit proper translation of mRNA,and stop codons. The term “control sequences” is intended to include, ata minimum, components whose presence can influence expression, and canalso include additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences. Expressioncontrol sequences can include a promoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see for example,Bitter et al., Methods in Enzymology 153:516-544, 1987). For example,when cloning in bacterial systems, inducible promoters such as pL ofbacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) andthe like may be used. In one embodiment, when cloning in mammalian cellsystems, promoters derived from the genome of mammalian cells (such asmetallothionein promoter) or from mammalian viruses (such as theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences.

A polynucleotide can be inserted into an expression vector, including aviral vector, containing a promoter sequence, which facilitates theefficient transcription of the inserted genetic sequence of the host.The expression vector typically contains an origin of replication, apromoter, as well as specific nucleic acid sequences that allowphenotypic selection of the transformed cells.

Heterologous: A heterologous polypeptide (such as a heterologousantigen) or polynucleotide refers to a polypeptide or polynucleotidederived from a different source or species. In some embodiments herein,the heterologous sequence is from a different genetic source, such as avirus or other organism, than the second sequence. In particularexamples, the heterologous sequence is a nucleic acid sequence encodinga tumor antigen or a pathogen-specific antigen.

Immunogenic (or antigenic) peptide: A peptide which comprises anallele-specific motif or other sequence, such as an N-terminal repeat,such that the peptide will bind an MHC molecule and induce a cytotoxic Tlymphocyte (“CTL”) response, or a B cell response (for example antibodyproduction) against the antigen from which the immunogenic peptide isderived. In one embodiment, immunogenic peptides are identified usingsequence motifs or other methods, such as neural net or polynomialdeterminations known in the art. Typically, algorithms are used todetermine the “binding threshold” of peptides to select those withscores that give them a high probability of binding at a certainaffinity and will be immunogenic. The algorithms are based either on theeffects on MHC binding of a particular amino acid at a particularposition, the effects on antibody binding of a particular amino acid ata particular position, or the effects on binding of a particularsubstitution in a motif-containing peptide. Within the context of animmunogenic peptide, a “conserved residue” is one which appears in asignificantly higher frequency than would be expected by randomdistribution at a particular position in a peptide. In one embodiment, aconserved residue is one where the MHC structure may provide a contactpoint with the immunogenic peptide.

Immune response: A change in immunity, for example a response of a cellof the immune system, such as a B-cell, T cell, macrophage, monocyte, orpolymorphonucleocyte, to an immunogenic agent in a subject. The responsecan be specific for a particular antigen (an “antigen-specificresponse”). In a particular example, an immune response is a T cellresponse, such as a CD4+ response or a CD8+ response. In anotherexample, the response is a B-cell response, and results in theproduction of specific antibodies to the immunogenic agent. In someexamples, such an immune response provides protection for the subjectfrom the immunogenic agent or the source of the immunogenic agent. Forexample, the response can treat a subject having a tumor, for example byinterfering with the metastasis of the tumor or reducing the number orsize of a tumor. In another example, the immune response can treat asubject with an infectious disease. An immune response can be active andinvolve stimulation of the subject's immune system, or be a responsethat results from passively acquired immunity. A “repeatedly stimulated”immune response is a long-term immune response resulting from theperiodic and repetitive stimulation of the immune system by the repeatedproduction of an antigen within a host. In some examples, an increasedor enhanced immune response is an increase in the ability of a subjectto fight off a disease, such as a tumor or infectious disease.

Immunity: The state of being able to mount a protective response uponexposure to an immunogenic agent. Protective responses can beantibody-mediated or immune cell-mediated, and can be directed toward aparticular pathogen or tumor antigen. Immunity can be acquired actively(such as by exposure to an immunogenic agent, either naturally or in apharmaceutical composition) or passively (such as by administration ofantibodies or in vitro stimulated and expanded T cells). In someembodiments disclosed herein, immunity is acquired by administration(such as by intraperitoneal or intravenous administration) of arecombinant CMV vector that is expressing a particular antigen, such asa pathogen-specific antigen or a tumor antigen.

Infectious disease: A disease caused by a pathogen, such as a fungus,parasite, protozoan, bacterium or virus.

Inhibiting or treating a disease. Inhibiting the full development orrecurrence of a disease or condition. “Treatment” refers to atherapeutic intervention that ameliorates a sign or symptom of a diseaseor pathological condition after it has begun to develop. The term“ameliorating,” with reference to a disease or pathological condition,refers to any observable beneficial effect of the treatment. Thebeneficial effect can be evidenced, for example, by a delayed onset orrecurrence of clinical symptoms of the disease in a susceptible subject,a reduction in severity of some or all clinical symptoms of the disease,a slower progression of the disease, a reduction in the number ofmetastases (if the disease is cancer), an decrease in titer of apathogen (if the disease is an infectious disease), an improvement inthe overall health or well-being of the subject, or by other parameterswell known in the art that are specific to the particular disease. A“prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs for thepurpose of decreasing the risk of developing pathology.

Isolated or non-naturally occurring: An “isolated” or “non-naturallyoccurring” biological component (such as a nucleic acid molecule,protein or organelle) has been substantially separated or purified awayfrom at least one other biological components in the cell of theorganism in which the component naturally occurs, e.g., otherchromosomal and extra-chromosomal DNA and RNA, proteins and organelles.Nucleic acids and proteins that are “non-naturally occurring” or havebeen “isolated” include nucleic acids and proteins purified by standardpurification methods. The term also embraces nucleic acids and proteinsprepared by recombinant expression in a host cell as well as chemicallysynthesized nucleic acids.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Open reading frame (ORF): A series of nucleotide triplets (codons)coding for amino acids without any internal termination codons. Thesesequences are usually translatable into a peptide.

Pathogen: A biological agent that causes disease or illness to its host.Pathogens include, for example, bacteria, viruses, fungi and protozoa.Pathogens are also referred to as infectious agents.

Examples of pathogenic viruses include, but are not limited to those inthe following virus families: Retroviridae (for example, humanimmunodeficiency virus (HIV), human T-cell leukemia viruses;Picornaviridae (for example, polio virus, hepatitis A virus, hepatitis Cvirus, enteroviruses, human coxsackie viruses, rhinoviruses,echoviruses, foot-and-mouth disease virus); Caliciviridae (such asstrains that cause gastroenteritis, including Norwalk virus);Togaviridae (for example, alphaviruses (including chikungunya virus,equine encephalitis viruses, Simliki Forest virus, Sindbis virus, RossRiver virus), rubella viruses); Flaviridae (for example, dengue viruses,yellow fever viruses, West Nile virus, St. Louis encephalitis virus,Japanese encephalitis virus, Powassan virus and other encephalitisviruses); Coronaviridae (for example, coronaviruses, severe acuterespiratory syndrome (SARS) virus; Rhabdoviridae (for example, vesicularstomatitis viruses, rabies viruses); Filoviridae (for example, Ebolavirus, Marburg virus); Paramyxoviridae (for example, parainfluenzaviruses, mumps virus, measles virus, respiratory syncytial virus);Orthomyxoviridae (for example, influenza viruses); Bunyaviridae (forexample, Hantaan viruses, Sin Nombre virus, Rift Valley fever virus,bunya viruses, phleboviruses and Nairo viruses); Arenaviridae (such asLassa fever virus and other hemorrhagic fever viruses, Machupo virus,Junin virus); Reoviridae (e.g., reoviruses, orbiviurses, rotaviruses);Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses,BK-virus); Adenoviridae (adenoviruses); Herpesviridae (herpes simplexvirus (HSV)-1 and HSV-2; cytomegalovirus; Epstein-Barr virus; varicellazoster virus; and other herpes viruses, including HSV-6); Poxyiridae(variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (suchas African swine fever virus); Astroviridae; and unclassified viruses(for example, the etiological agents of spongiform encephalopathies, theagent of delta hepatitis (thought to be a defective satellite ofhepatitis B virus).

Examples of bacterial pathogens include, but are not limited to:Helicobacter pylori, Escherichia coli, Vibrio cholerae, Boreliaburgdorferi, Legionella pneumophilia, Mycobacteria sps (such as. M.tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae),Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis,Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus),Streptococcus agalactiae (Group B Streptococcus), Streptococcus(viridans group), Streptococcus faecalis, Streptococcus bovis,Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenicCampylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillusanthracis, corynebacterium diphtheriae, corynebacterium sp.,Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridiumtetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturellamultocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillusmoniliformis, Treponema pallidium, Treponema pertenue, Leptospira,Bordetella pertussis, Shigella flexnerii, Shigella dysenteriae andActinomyces israelli.

Examples of fungal pathogens include, but are not limited to:Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis,Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.

Examples of pathogens such as parasitic/protozoan pathogens include, butare not limited to: Plasmodium falciparum, Plasmodium vivax, Trypanosomacruzi and Toxoplasma gondii. The invention relates to a parasite thatmay be a protozoan organism or organisms which cause diseases thatinclude, but not limited to, Acanthamoeba, Babesiosis, Balantidiasis,Blastocystosis, Coccidia, Dientamoebiasis, Amoebiasis, Giardia,Isosporiasis, Leishmaniasis, Primary amoebic meningoencephalitis (PAM),Malaria, Rhinosporidiosis, Toxoplasmosis—Parasitic pneumonia,Trichomoniasis, Sleeping sickness and Chagas disease. The parasite maybe a helminth organism or worm or organisms which cause diseases thatinclude, but not limited to, Ancylostomiasis/Hookworm, Anisakiasis,Roundworm—Parasitic pneumonia, Roundworm—Baylisascariasis,Tapeworm—Tapeworm infection, Clonorchiasis, Dioctophyme renalisinfection, Diphyllobothriasis—tapeworm, Guinea worm—Dracunculiasis,Echinococcosis—tapeworm, Pinworm—Enterobiasis, Liver fluke—Fasciolosis,Fasciolopsiasis—intestinal fluke, Gnathostomiasis, Hymenolepiasis, Loaboa filariasis, Calabar swellings, Mansonelliasis, Filariasis,Metagonimiasis—intestinal fluke, River blindness, Chinese Liver Fluke,Paragonimiasis, Lung Fluke, Schistosomiasis—bilharzia, bilharziosis orsnail fever (all types), intestinal schistosomiasis, urinaryschistosomiasis, Schistosomiasis by Schistosoma japonicum, Asianintestinal schistosomiasis, Sparganosis, Strongyloidiasis—Parasiticpneumonia, Beef tapeworm, Pork tapeworm, Toxocariasis, Trichinosis,Swimmer's itch, Whipworm and Elephantiasis Lymphatic filariasis. Theparasite may be an organism or organisms which cause diseases thatinclude, but not limited to, parasitic worm, Halzoun Syndrome, Myiasis,Chigoe flea, Human Botfly and Candiru. The parasite may be anectoparasite or organisms which cause diseases that include, but are notlimited to, Bedbug, Head louse—Pediculosis, Body louse—Pediculosis, Crablouse—Pediculosis, Demodex—Demodicosis, Scabies, Screwworm andCochliomyia.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful with this disclosure are conventional. Martin,Remington's Pharmaceutical Sciences, published by Mack Publishing Co.,Easton, Pa., 19th Edition, 1995, describes compositions and formulationssuitable for pharmaceutical delivery of the nucleotides and proteinsherein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Plaque forming units (PFU): A measure of virus dose or titer, determinedby its ability to form plaques on a permissive cell line.

Polypeptide: A polymer in which the monomers are amino acid residuesthat are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used, the L-isomers being preferred. The term polypeptide orprotein as used herein encompasses any amino acid sequence and includesmodified sequences such as glycoproteins. The term polypeptide isspecifically intended to cover naturally occurring proteins, as well asthose that are recombinantly or synthetically produced.

The term polypeptide fragment refers to a portion of a polypeptide thatexhibits at least one useful epitope. The phrase “functional fragment(s)of a polypeptide” refers to all fragments of a polypeptide that retainan activity, or a measurable portion of an activity, of the polypeptidefrom which the fragment is derived. Fragments, for example, can vary insize from a polypeptide fragment as small as an epitope capable ofbinding an antibody molecule to a large polypeptide capable ofparticipating in the characteristic induction or programming ofphenotypic changes within a cell. An epitope is a region of apolypeptide capable of binding an immunoglobulin generated in responseto contact with an antigen.

Conservative amino acid substitution tables providing functionallysimilar amino acids are well known to one of ordinary skill in the art.The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine I, Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

In some circumstances, variations in the cDNA sequence that result inamino acid changes, whether conservative or not, are minimized in orderto preserve the functional and immunologic identity of the encodedprotein. The immunologic identity of the protein may be assessed bydetermining whether it is recognized by an antibody; a variant that isrecognized by such an antibody is immunologically conserved. Any cDNAsequence variant will preferably introduce no more than twenty, andpreferably fewer than ten amino acid substitutions into the encodedpolypeptide. Variant amino acid sequences may, for example, be 80%, 90%,or even 95% or 98% identical to the native amino acid sequence. Programsand algorithms for determining percentage identity can be found at theNCBI website.

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purified proteinpreparation is one in which the protein referred to is more pure thanthe protein in its natural environment within a cell or within aproduction reaction chamber (as appropriate).

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination can be accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques.

Replication-deficient: As used herein, a replication-deficient CMV is avirus that once in a host cell, cannot undergo viral replication, or issignificantly limited in its ability to replicate its genome and thusproduce progeny virions. In other examples, replication-deficientviruses are dissemination-deficient, i.e. they are capable ofreplicating their genomes, but unable to infect another cell eitherbecause virus particles are not released from the infected cell orbecause non-infectious viral particles are released. In other examples,replication-deficient viruses are spread-deficient, i.e. infectiousvirus is not secreted from the infected host are therefore the virus isincapable to spread from host to host. In some embodiments, areplication-deficient CMV is a CMV comprising a deletion in one or moregenes essential for viral replication (“essential genes”) or requiredfor optimal replication (“augmenting genes”). CMV essential andaugmenting genes have been described in the art and are disclosedherein. In particular examples, the CMV essential or augmenting gene isUL82, UL94, UL32, UL99, UL115 or UL44 (or the RhCMV homolog thereof).

Sample or biological sample: A biological specimen obtained from asubject, such as a cell, fluid of tissue sample. In some cases,biological samples contain genomic DNA, RNA (including mRNA andmicroRNA), protein, or combinations thereof. Examples of samplesinclude, but are not limited to, saliva, blood, serum, urine, spinalfluid, tissue biopsy, surgical specimen, cells (such as PBMCs, whiteblood cells, lymphocytes, or other cells of the immune system) andautopsy material.

Sequence identity: The similarity between two nucleic acid sequences, ortwo amino acid sequences, is expressed in terms of the similaritybetween the sequences, otherwise referred to as sequence identity.Sequence identity is frequently measured in terms of percentage identity(or similarity or homology); the higher the percentage, the more similarthe two sequences are.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman (Adv. Appl. Math. 2: 482, 1981); Needleman and Wunsch (J.Mol. Biol. 48: 443, 1970); Pearson and Lipman (PNAS USA 85: 2444, 1988);Higgins and Sharp (Gene, 73: 237-244, 1988); Higgins and Sharp (CABIOS5: 151-153, 1989); Corpet et al. (Nuc. Acids Res. 16: 10881-10890,1988); Huang et al. (Comp. Appls Biosci. 8: 155-165, 1992); and Pearsonet al. (Meth. Mol. Biol. 24: 307-31, 1994). Altschul et al. (NatureGenet., 6: 119-129, 1994) presents a detailed consideration of sequencealignment methods and homology calculations. The alignment tools ALIGN(Myers and Miller, CABIOS 4:11-17, 1989) or LFASTA (Pearson and Lipman,1988) may be used to perform sequence comparisons (Internet Program©1996, W. R. Pearson and the University of Virginia, fasta20u63 version2.0u63, release date December 1996). ALIGN compares entire sequencesagainst one another, while LFASTA compares regions of local similarity.These alignment tools and their respective tutorials are available onthe Internet at the NCSA Website. Alternatively, for comparisons ofamino acid sequences of greater than about 30 amino acids, the Blast 2sequences function can be employed using the default BLOSUM62 matrix setto default parameters, (gap existence cost of 11, and a per residue gapcost of 1). When aligning short peptides (fewer than around 30 aminoacids), the alignment should be performed using the Blast 2 sequencesfunction, employing the PAM30 matrix set to default parameters (open gap9, extension gap 1 penalties). The BLAST sequence comparison system isavailable, for instance, from the NCBI web site; see also Altschul etal., J. Mol. Biol. 215:403-410, 1990; Gish. & States, Nature Genet.3:266-272, 1993; Madden et al. Meth. Enzymol. 266:131-141, 1996;Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; and Zhang &Madden, Genome Res. 7:649-656, 1997.

Orthologs of proteins are typically characterized by possession ofgreater than 75% sequence identity counted over the full-lengthalignment with the amino acid sequence of specific protein using ALIGNset to default parameters. Proteins with even greater similarity to areference sequence will show increasing percentage identities whenassessed by this method, such as at least 80%, at least 85%, at least90%, at least 92%, at least 95%, or at least 98% sequence identity. Inaddition, sequence identity can be compared over the full length ofparticular domains of the disclosed peptides.

When significantly less than the entire sequence is being compared forsequence identity, homologous sequences will typically possess at least80% sequence identity over short windows of 10-20 amino acids, and maypossess sequence identities of at least 85%, at least 90%, at least 95%,or at least 99% depending on their similarity to the reference sequence.Sequence identity over such short windows can be determined usingLFASTA; methods are described at the NCSA Website. One of skill in theart will appreciate that these sequence identity ranges are provided forguidance only; it is entirely possible that strongly significanthomologs could be obtained that fall outside of the ranges provided.

An alternative indication that two nucleic acid molecules are closelyrelated is that the two molecules hybridize to each other understringent conditions. Stringent conditions are sequence-dependent andare different under different environmental parameters. Generally,stringent conditions are selected to be about 5° C. to 20° C. lower thanthe thermal melting point I for the specific sequence at a defined ionicstrength and pH. The Tm is the temperature (under defined ionic strengthand pH) at which 50% of the target sequence hybridizes to a perfectlymatched probe. Conditions for nucleic acid hybridization and calculationof stringencies can be found in Sambrook et al. (In Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, N. Y., 1989) and Tijssen(Laboratory Techniques in Biochemistry and Molecular Biology Part I, Ch.2, Elsevier, New York, 1993).

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing nucleic acidsequences. Generally, the temperature of hybridization and the ionicstrength (especially the Na+ concentration) of the hybridization bufferwill determine the stringency of hybridization, though waste times alsoinfluence stringency. Calculations regarding hybridization conditionsrequired for attaining particular degrees of stringency are discussed bySambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed.,vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, chapters 9 and 11, herein incorporated by reference. Thefollowing is an exemplary set of hybridization conditions:

Very High Stringency (Detects Sequences that Share 90% Identity)

Hybridization: 5×SSC at 65° C. for 16 hours

Wash twice: 2×SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5×SSC at 65° C. for 20 minutes each

High Stringency (Detects Sequences that Share 80% Identity or Greater)

Hybridization: 5×-6×SSC at 65° C.-70° C. for 16-20 hours

Wash twice: 2×SSC at RT for 5-20 minutes each

Wash twice: 1×SSC at 55° C.-70° C. for 30 minutes each

Low Stringency (Detects Sequences that Share Greater than 50% Identity)

Hybridization: 6×SSC at RT to 55° C. for 16-20 hours

Wash at least twice: 2×-3×SSC at RT to 55° C. for 20-30 minutes each.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences, due to the degeneracyof the genetic code. It is understood that changes in nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid sequences that each encode substantially the same protein.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and non-human mammals. This term encompasses bothknown and unknown individuals, such that there is no requirement that aperson working with a sample from a subject know who the subject is, oreven from where the sample was acquired.

Tumor or cancer antigen: An antigen that can stimulate tumor-specificT-cell immune responses. Exemplary tumor antigens include, but are notlimited to, RAGE-1, tyrosinase, MAGE-1, MAGE-2, NY-ESO-1,Melan-A/MART-1, glycoprotein (gp) 75, gp100, beta-catenin, PRAME, MUM-1,WT-1, CEA, and PR-1. Additional tumor antigens are known in the art (forexample see Novellino et al., Cancer Immunol. Immunother. 54(3):187-207,2005) and are described below (see Table 2). Cancer antigen and tumorantigen are used interchangeably herein. The antigens may be related tocancers that include, but are not limited to, Acute lymphoblasticleukemia; Acute myeloid leukemia; Adrenocortical carcinoma; AIDS-relatedcancers; AIDS-related lymphoma; Anal cancer; Appendix cancer;Astrocytoma, childhood cerebellar or cerebral; Basal cell carcinoma;Bile duct cancer, extrahepatic; Bladder cancer; Bone cancer,Osteosarcoma/Malignant fibrous histiocytoma; Brainstem glioma; Braintumor; Brain tumor, cerebellar astrocytoma; Brain tumor, cerebralastrocytoma/malignant glioma; Brain tumor, ependymoma; Brain tumor,medulloblastoma; Brain tumor, supratentorial primitive neuroectodermaltumors; Brain tumor, visual pathway and hypothalamic glioma; Breastcancer; Bronchial adenomas/carcinoids; Burkitt lymphoma; Carcinoidtumor, childhood; Carcinoid tumor, gastrointestinal; Carcinoma ofunknown primary; Central nervous system lymphoma, primary; Cerebellarastrocytoma, childhood; Cerebral astrocytoma/Malignant glioma,childhood; Cervical cancer; Childhood cancers; Chronic lymphocyticleukemia; Chronic myelogenous leukemia; Chronic myeloproliferativedisorders; Colon Cancer; Cutaneous T-cell lymphoma; Desmoplastic smallround cell tumor; Endometrial cancer; Ependymoma; Esophageal cancer;Ewing's sarcoma in the Ewing family of tumors; Extracranial germ celltumor, Childhood; Extragonadal Germ cell tumor; Extrahepatic bile ductcancer; Eye Cancer, Intraocular melanoma; Eye Cancer, Retinoblastoma;Gallbladder cancer; Gastric (Stomach) cancer; Gastrointestinal CarcinoidTumor; Gastrointestinal stromal tumor (GIST); Germ cell tumor:extracranial, extragonadal, or ovarian; Gestational trophoblastic tumor;Glioma of the brain stem; Glioma, Childhood Cerebral Astrocytoma;Glioma, Childhood Visual Pathway and Hypothalamic; Gastric carcinoid;Hairy cell leukemia; Head and neck cancer; Heart cancer; Hepatocellular(liver) cancer; Hodgkin lymphoma; Hypopharyngeal cancer; Hypothalamicand visual pathway glioma, childhood; Intraocular Melanoma; Islet CellCarcinoma (Endocrine Pancreas); Kaposi sarcoma; Kidney cancer (renalcell cancer); Laryngeal Cancer; Leukemias; Leukemia, acute lymphoblastic(also called acute lymphocytic leukemia); Leukemia, acute myeloid (alsocalled acute myelogenous leukemia); Leukemia, chronic lymphocytic (alsocalled chronic lymphocytic leukemia); Leukemia, chronic myelogenous(also called chronic myeloid leukemia); Leukemia, hairy cell; Lip andOral Cavity Cancer; Liver Cancer (Primary); Lung Cancer, Non-Small Cell;Lung Cancer, Small Cell; Lymphomas; Lymphoma, AIDS-related; Lymphoma,Burkitt; Lymphoma, cutaneous T-Cell; Lymphoma, Hodgkin; Lymphomas,Non-Hodgkin (an old classification of all lymphomas except Hodgkin's);Lymphoma, Primary Central Nervous System; Marcus Whittle, DeadlyDisease; Macroglobulinemia, Waldenström; Malignant Fibrous Histiocytomaof Bone/Osteosarcoma; Medulloblastoma, Childhood; Melanoma; Melanoma,Intraocular (Eye); Merkel Cell Carcinoma; Mesothelioma, Adult Malignant;Mesothelioma, Childhood; Metastatic Squamous Neck Cancer with OccultPrimary; Mouth Cancer; Multiple Endocrine Neoplasia Syndrome, Childhood;Multiple Myeloma/Plasma Cell Neoplasm; Mycosis Fungoides;Myelodysplastic Syndromes; Myelodysplastic/Myeloproliferative Diseases;Myelogenous Leukemia, Chronic; Myeloid Leukemia, Adult Acute; MyeloidLeukemia, Childhood Acute; Myeloma, Multiple (Cancer of theBone-Marrow); Myeloproliferative Disorders, Chronic; Nasal cavity andparanasal sinus cancer; Nasopharyngeal carcinoma; Neuroblastoma;Non-Hodgkin lymphoma; Non-small cell lung cancer; Oral Cancer;Oropharyngeal cancer; Osteosarcoma/malignant fibrous histiocytoma ofbone; Ovarian cancer; Ovarian epithelial cancer (Surfaceepithelial-stromal tumor); Ovarian germ cell tumor; Ovarian lowmalignant potential tumor; Pancreatic cancer; Pancreatic cancer, isletcell; Paranasal sinus and nasal cavity cancer; Parathyroid cancer;Penile cancer; Pharyngeal cancer; Pheochromocytoma; Pineal astrocytoma;Pineal germinoma; Pineoblastoma and supratentorial primitiveneuroectodermal tumors, childhood; Pituitary adenoma; Plasma cellneoplasia/Multiple myeloma; Pleuropulmonary blastoma; Primary centralnervous system lymphoma; Prostate cancer; Rectal cancer; Renal cellcarcinoma (kidney cancer); Renal pelvis and ureter, transitional cellcancer; Retinoblastoma; Rhabdomyosarcoma, childhood; Salivary glandcancer; Sarcoma, Ewing family of tumors; Sarcoma, Kaposi; Sarcoma, softtissue; Sarcoma, uterine; Sézary syndrome; Skin cancer (nonmelanoma);Skin cancer (melanoma); Skin carcinoma, Merkel cell; Small cell lungcancer; Small intestine cancer; Soft tissue sarcoma; Squamous cellcarcinoma—see Skin cancer (nonmelanoma); Squamous neck cancer withoccult primary, metastatic; Stomach cancer; Supratentorial primitiveneuroectodermal tumor, childhood; T-Cell lymphoma, cutaneous (MycosisFungoides and Sézary syndrome); Testicular cancer; Throat cancer;Thymoma, childhood; Thymoma and Thymic carcinoma; Thyroid cancer;Thyroid cancer, childhood; Transitional cell cancer of the renal pelvisand ureter; Trophoblastic tumor, gestational; Unknown primary site,carcinoma of, adult; Unknown primary site, cancer of, childhood; Ureterand renal pelvis, transitional cell cancer; Urethral cancer; Uterinecancer, endometrial; Uterine sarcoma; Vaginal cancer; Visual pathway andhypothalamic glioma, childhood; Vulvar cancer; Waldenströmmacroglobulinemia and Wilms tumor (kidney cancer), childhood.

Under conditions sufficient for/to: A phrase that is used to describeany environment that permits the desired activity.

Vaccine: An immunogenic composition that can be administered to amammal, such as a human, to confer immunity, such as active immunity, toa disease or other pathological condition. Vaccines can be usedprophylactically or therapeutically. Thus, vaccines can be used reducethe likelihood of developing a disease (such as a tumor or infection) orto reduce the severity of symptoms of a disease or condition, limit theprogression of the disease or condition (such as a tumor or infection),or limit the recurrence of a disease or condition. In particularembodiments, a vaccine is a recombinant CMV (such as a recombinant HCMVor recombinant RhCMV) expressing a heterologous antigen, such as apathogen-specific antigen or a tumor antigen.

Vector: Nucleic acid molecules of particular sequence can beincorporated into a vector that is then introduced into a host cell,thereby producing a transformed host cell. A vector may include nucleicacid sequences that permit it to replicate in a host cell, such as anorigin of replication. A vector may also include one or more selectablemarker genes and other genetic elements known in the art, includingpromoter elements that direct nucleic acid expression. Vectors can beviral vectors, such as CMV vectors. Viral vectors may be constructedfrom wild type or attenuated virus, including replication deficientvirus. Vectors can also be non-viral vectors, including any plasmidknown to the art.

Virus: Microscopic infectious organism that reproduces inside livingcells. A virus consists essentially of a core of nucleic acid (the viralgenome) surrounded by a protein coat (capsid), and has the ability toreplicate only inside a living cell. “Viral replication” is theproduction of additional virus particles by the occurrence of at leastone viral life cycle. A virus may subvert the host cells' normalfunctions, causing the cell to behave in a manner determined by thevirus. For example, a viral infection may result in a cell producing acytokine, or responding to a cytokine, when the uninfected cell does notnormally do so.

In a “lytic” or “acute” viral infection, the viral genome is replicatedand expressed, producing the polypeptides necessary for production ofthe viral capsid. Mature viral particles exit the host cell, resultingin cell lysis. Particular viral species can alternatively enter into a“‘lysogenic” or “latent” infection. In the establishment of latency, theviral genome is replicated, but capsid proteins are not produced andassembled into viral particles.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Hence “comprisingA or B” means including A, or B, or A and B. It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

Described herein are recombinant RhCMV and HCMV vectors encodingheterologous antigens, such as pathogen-specific antigens or tumorantigens. The recombinant vectors disclosed herein elicit and maintainhigh level cellular and humoral immune responses specific for theheterologous antigen. Thus, the disclosed CMV vectors are suitable foruse as vaccines to treat or prevent infectious disease and cancer. Alsodisclosed are recombinant RhCMV and HCMV vectors lacking at least oneessential or augmenting gene (replication-deficient viruses).Replication-deficient CMVs can include a heterologous antigen, such as apathogen-specific antigen or a tumor antigen, and thus can be used asvaccines to treat or prevent the corresponding infection or cancer. Inother cases, the replication-deficient CMVs, which are attenuated, lacka heterologous antigen and can be used as a vaccine against CMV.

Thus, provided herein are recombinant RhCMV or HCMV vectors comprising anucleic acid sequence encoding a heterologous antigen. In someembodiments, the heterologous antigen is a pathogen-specific antigen. Inother embodiments, the heterologous antigen is a tumor antigen.

In some embodiments, the pathogen-specific antigen is a viral antigen.The viral antigen can be from any virus that is known to be pathogenic,or against which it is desirable to elicit an immune response. In someexamples, the viral antigen is an antigen from influenza virus,monkeypox virus, West Nile virus, Chikungunya virus, Ebola virus,hepatitis C virus, poliovirus, dengue virus serotype 1, dengue virusserotype 2, dengue virus serotype 3 or dengue virus serotype 4,Papillomavirus, SIV, HIV, HCMV, Kaposi's sarcoma associated herpesvirus,varcella zoster virus, Epstein-barr virus, Herpes simplex 1 virus andHerpes simplex 2 virus. Specific antigens from these and other virusesare well known in the art. Thus, a suitable antigen can be selected byone of ordinary skill in the art.

In particular examples, the influenza virus antigen is hemagglutinin orneuraminidase, or an epitope or antigenic fragment thereof; themonkeypox virus antigen is A35R, or an epitope or antigenic fragmentthereof; the West Nile virus antigen is capsid (C), membrane (prM/M),envelope (E), NS1, NS2A, NS2B, NS3, NS4A, NS4B or NS5, or an epitope ofantigenic fragment thereof; the Chikungunya virus antigen is capsid (C),envelope glycoprotein 1 (E1), envelope glycoprotein 2 (E2), envelopeglycoprotein 3 (E3) or non-structural protein 1 (NSP1), or an epitope orantigenic fragment thereof; the Ebola virus antigen is nucleoprotein(NP), or an epitope or antigenic fragment thereof; the hepatitis C virusantigen is core, E1, E2, NS2, NS3, NS4 or NS5, or an epitope orantigenic fragment thereof; the poliovirus antigen is VP1, or an epitopeor antigenic fragment thereof; or the dengue virus antigen is capsid(C), membrane (prM/M), envelope (E), NS1, NS2A, NS2B, NS3, NS4A, NS4B orNS5, or an epitope or antigenic fragment thereof.

In some embodiments in which the recombinant RhCMV or HCMV vector isreplication-deficient, the vector does not include a heterologousantigen. Such a recombinant vector, which is attenuated, can be used totreat or prevent infection with CMV.

In some embodiments, the pathogen-specific antigen is a bacterialantigen. The bacterial antigen can be from any type of bacteria that isknown to be pathogenic, or against which it is desirable to elicit animmune response. In some embodiments, the bacterial antigen is fromMycobacterium tuberculosis, the causative agent of Tuberculosis. In someembodiments, the bacterial antigen is from Clostridium tetani, thecausative agent of tetanus. Specific antigens from Clostridium tetaniare well known in the art. Thus, a suitable antigen from Clostridiumtetani can be selected by one of ordinary skill in the art. Inparticular examples, the antigen from Clostridium tetani is tetanusC.

In some embodiments, the pathogen-specific antigen is a fungal antigen.The fungal antigen can be from any fungus that is known to bepathogenic, or against which it is desirable to elicit an immuneresponse.

In some embodiments, the pathogen-specific antigen is a protozoanantigen. The protozoan antigen can be from any protozoan that is knownto be pathogenic, or against which it is desirable to elicit an immuneresponse. In some examples, the protozoan antigen is an antigen from aPlasmodium species, such as Plasmodium falciparum, Plasmodium vivax,Plasmodium ovale or Plasmodium malariae. Specific antigens fromPlasmodium species are well known in the art. Thus, a suitable antigencan be selected by one of ordinary skill in the art. In particularexamples, the antigen from Plasmodium is a pre-erythrocytic antigen,such as CSP or SSP2, or an erythrocytic antigen, such as AMA1 or MSP1.

In some embodiments, the tumor antigen is an antigen expressed by asolid tumor. In other embodiments, the tumor antigen is an antigenexpressed by a hematological cancer. Tumor antigens are well known inthe art and a non-limiting list of tumor antigens is provided herein.

The RhCMV or HCMV vectors can be derived from any RhCMV or HCMV virusisolate, strain or variant (such as a clinical isolate or laboratorystrain). In some embodiments, the RhCMV is Cercopithecine herpesvirus 8.In particular examples, the RhCMV comprises the nucleotide sequence ofSEQ ID NO: 1, or a nucleotide sequence that is at least 80%, at least85%, at least 90%, at least 95% or at least 99% identical to SEQ IDNO: 1. The RhCMV vector can also be BAC encoding the RhCMV genome. Insome embodiments, recombinant RhCMV vector includes a deletion of one ormore genes encoding an immunomodulatory protein. In some examples, thedeletion comprises a deletion of Rh182-189 or Rh158-166, or both.

In some embodiments, the HCMV comprises the AD169 lab strain, wild-typestrain Merlin, Towne BAC HCMV isolate, Toledo-BAC HCMV isolate, TR-BACHCMV isolate, FIX-BAC HCMV isolate, AD169-BAC HCMV isolate or HCMVstrain Davis. In particular examples, the HCMV vector comprises thenucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. Incertain embodiments, a variant of a HCMV or RhCMV vector can be used,for example, a variant that is at least 80%, at least 85%, at least 90%,at least 95% or at least 99% identical to any one of SEQ ID NOs: 2-9,but that retains the capacity to function as a vector.

Recombinant RhCMV and CMV vectors are publicly available and/or havebeen previously described. For example, several CMV vectors areavailable from the American Type Culture Collection (Manassas, Va.),including HCMV AD169 (ATCC VR-538), HCMV Towne (ATCC VR-977) and HCMVDavis (ATCC VR-807). HCMV Toledo strain has also been described (seeQuinnan et al., Ann Intern Med 101: 478-83, 1984). Recombinant HCMV andRhCMV are also described in, for example, U. S. Patent ApplicationPublication No. 2009/029755 and PCT Publication No. WO 2006/031264,which is incorporated herein by reference.

In some embodiments, the recombinant RhCMV or HCMV vector comprises adeletion in a RhCMV or HCMV gene that is essential for or augmentsreplication. CMV essential genes and augmenting have been well describedin the art (see, for example, Dunn et al., Proc. Natl. Acad. Sci. USA100(24):14223-14228, 2003; and Dong et al., Proc. Natl. Acad. Sci. USA100(21):12396-12401, 2003). Essential CMV genes include, but are notlimited to, UL32, UL34, UL37, UL44, UL46, UL48, UL48.5, UL49, UL50,UL51, UL52, UL53, UL54, UL55, UL56, UL57, UL60, UL61, UL70, UL71, UL73,UL75, UL76, UL77, UL79, UL80, UL82, UL84, UL85, UL86, UL87, UL89, UL90,UL91, UL92, UL93, UL94, UL95, UL96, UL98, UL99, UL100, UL102, UL104,UL105, UL115 and UL122. In some embodiments, the CMV essential oraugmenting gene is UL82, UL94, UL32, UL99, UL115 or UL44, or a homologthereof (i.e., the homologous gene in RhCMV). Other essential oraugmenting genes are known in the art and are described herein. Inparticular examples, the essential gene is UL82, or a homolog thereof.In some embodiments, the recombinant RhCMV and HCMV vectors do notinclude a heterologous antigen. In other embodiments, the recombinantRhCMV or HCMV vector having a deletion in an essential or augmentinggene includes a nucleic acid sequence encoding a heterologous antigen,such as a pathogen-specific antigen or a tumor antigen. Compositionscomprising recombinant RhCMV or HCMV vectors and a pharmaceuticallyacceptable carrier also are provided. Such vectors and compositions canbe used, for example, in a method of treating a subject with aninfectious disease, or at risk of becoming infected with an infectiousdisease, or with cancer, or at risk of developing cancer. CMV vectorshaving a deletion of at least one essential or augmenting gene aregenerally attenuated and thus can be used as vaccines for the treatmentor prevention of CMV (in which case, the recombinant vector does notencode a heterologous antigen).

In some embodiments, the recombinant RhCMV or HCMV vectors comprise asuicide or safety means to prevent further replication of the virus. Forexample, the recombinant CMV vectors can include LoxP sites flanking anessential gene or region of the RhCMV or HCMV genome (essential CMVgenes are listed above and are known in the art), as well as the codingsequence for Cre-recombinase. Cre-recombinase is generally under thecontrol of an inducible promoter to regulate expression of Cre, therebycontrolling removal of the essential gene and inhibition of viralreplication. In particular examples, Cre is a Tet-regulated Cre andexpression of Cre is controlled by the presence of Dox.

Also provided are compositions comprising a recombinant RhCMV or HCMVvector disclosed herein and a pharmaceutically acceptable carrier.

Further provided is a method of treating a subject with an infectiousdisease, or at risk of becoming infected with an infectious disease, orwith cancer, or at risk of developing cancer, by selecting a subject inneed of treatment and administering to the subject a recombinant RhCMVor HCMV vector, or composition thereof, disclosed herein. In someembodiments, selecting a subject in need of treatment includes selectinga subject diagnosed with an infectious disease, such as a viral disease,a bacterial disease, a fungal disease or a protozoan disease. In otherembodiments, selecting a subject in need of treatment includes selectinga subject that has been exposed or is likely to be exposed to aninfectious agent. In other embodiments, selecting a subject in need oftreatment includes selecting a subject diagnosed with cancer. In otherembodiments, selecting a subject in need of treatment includes selectinga subject that is at risk of developing cancer, such as a subject thathas been exposed to a carcinogen, a subject that has cancer associatedgenetic mutations or a subject that has previously had cancer.

In some embodiments of the methods, the infectious disease is influenzavirus infection and the recombinant RhCMV or HCMV vector encodes anantigen from influenza virus. In other embodiments, the infectiousdisease is monkeypox virus infection and the recombinant RhCMV or HCMVvector encodes an antigen from monkeypox virus. In other embodiments,the infectious disease is West Nile virus infection and the recombinantRhCMV or HCMV vector encodes an antigen from West Nile virus. In otherembodiments, the infectious disease is Chikungunya virus infection andthe recombinant RhCMV or HCMV vector encodes an antigen from Chikungunyavirus. In other embodiments, the infectious disease is Ebola virusinfection and the recombinant RhCMV or HCMV vector encodes an antigenfrom Ebola virus. In other embodiments, the infectious disease ishepatitis C virus infection and the recombinant RhCMV or HCMV vectorencodes an antigen from hepatitis C virus. In other embodiments, theinfectious disease is poliovirus infection and the recombinant RhCMV orHCMV vector encodes an antigen from poliovirus. In other embodiments,the infectious disease is dengue fever and the recombinant RhCMV or HCMVvector encodes an antigen from dengue virus serotype 1, dengue virusserotype 2, dengue virus serotype 3 or dengue virus serotype 4. In otherembodiments, the disease is Acquired immune deficiency syndrome (AIDS)or a Simian Immunodeficiency virus (SIV) infection and the recombinantRhCMV or HCMV vector encodes an antigen from HIV or SIV. In otherembodiments, the disease is or is related to skin warts, genital warts,cervical cancer or respiratory papillomatosis, and the recombinant RhCMVor HCMV vector encodes an antigen from HPV. In other embodiments, thedisease is Malaria and the recombinant RhCMV or HCMV vector encodes anantigen from eukaryotic protists of the genus Plasmodium. In otherembodiments, the disease is Ebola hemorrhagic fever and the recombinantRhCMV or HCMV vector encodes an antigen from the Ebola virus. In otherembodiments, the disease is Tuberculosis and the recombinant RhCMV orHCMV vector encodes an antigen from the Mycobacterium tuberculosis. Inother embodiments, the disease is Ebola hemorrhagic fever and therecombinant RhCMV or HCMV vector encodes an antigen from the Ebolavirus.

In other embodiments, the disease is a Herpes disease and therecombinant RhCMV or HCMV vector encodes an antigen from a Herpes virus.A herpes disease includes but is not limited to Genital Herpes, Chickenpox or Herpes Zoster (shingles), Infectious mononucleosis and Kaposi'ssarcoma. The recombinant RhCMV or HCMV vector encodes an antigen fromviruses that include but are not limited to Herpes simplex virus-1(HSV-1), Herpes simplex virus-2 (HSV-2), Varicella zorter virus (VZV),Epstein-Barr virus (EBV), Roseolovirus, and Kaposi's sarcoma-associatedherpesvirus (KSHV). In some embodiments of the methods, the infectiousdisease is CMV infection and the vector does not include a heterologousantigen.

In some embodiments of the methods, the cancer is a solid tumor and theRhCMV or HCMV vector encodes a tumor antigen from a solid tumor. Inother embodiments, the cancer is a hematological cancer and the RhCMV orHCMV vector encodes a tumor antigen from a hematological cancer.

Cytomegaloviruses (CMVs) comprise a distinct, widely distributedsubgroup of β-herpesviruses that share common growth characteristics,characteristic cytopathology, salivary gland tropism and a capacity toestablish persistent and latent infection. Among the largest and mostcomplex of known viruses, CMV virions range in size from 150-200 nm andinclude a double stranded DNA genome of 230 kb—capable of coding formore than 200 proteins. The β-herpes viruses in general, and the CMVs inspecific, are thought to have emerged prior to mammalian radiation, andtherefore, viral evolution has accompanied mammalian speciation suchthat each species has their own uniquely adapted CMV, and CMVrelatedness generally parallels species relatedness. Thus, the geneticsand biology of primate CMVs (human, chimp, RM) are considerably moreclosely related to each other than to rodent CMVs. In both humans indeveloping counties and in RM, CMV infection is essentially universalwith 90-100% of adults showing serologic evidence of infection (Vogel etal., Lab Anim Sci 44(1):25-30, 1994; Ho, Rev Infect Dis 12 Suppl7:S701-710, 1990). In affluent areas of developed countries, increasedhygiene has somewhat limited transmission with only 40-60% of the adultpopulation showing seroreactivity. For the vast majority of exposed(immunologically normal) subjects, either human or monkey, acuteinfection with CMV is completely asymptomatic (Zanghellini et al., JInfect Dis 180(3):702-707, 1999; Lockridge et al., J Virol73(11):9576-83, 1999); a small fraction of humans (<5%) experiencesymptomatic, but benign illness, and an even smaller fraction experiencea mononucleosis syndrome (CMV accounts for ˜8% of all cases ofmononucleosis) (Zanghellini et al., J Infect Dis 180(3):702-707, 1999).

After initial infection, CMV is shed for months to years in multiplebody fluids (saliva, tears, urine, genital secretions, breast milk), andtransmission generally involves mucosal exposure to such fluids, and notsurprisingly occurs in situations where such secretion ‘transfer’ iscommon (early childhood and adolescence). CMV persists in the hostindefinitely and viral shedding can occur years after exposure. CMV canmanifest latent infection of its target cells, and like other herpesviruses, this capability likely plays a role in this remarkablepersistence. However, unlike herpes simplex and varicella-zoster virus,the frequency of shedding (particularly in monkeys), the kinetics ofreactivation after transplantation, and the unique strength of the Tcell response suggest that foci of active CMV replication arefrequently, if not continuously, occurring somewhere in the infectedhost's body. Such active persistence implies a means for evading hostimmunity, and indeed, CMV has evolved diverse mechanisms formanipulating both innate and adaptive immune responses, including genesthat modulate/interfere with 1) Ag presentation and other majorhistocompatibility complex (MHC) protein function, 2) leukocytemigration, activation and cytokine responses, 3) Fc receptor function,and 4) host cell susceptibility to apoptosis induction (Mocarski, TrendsMicrobiol 10(7):332-329, 2002; Atalay et al., J Virol 76(17):8596-608,2002; Skaletskaya et al., Proc Natl Acad Sci USA 98(14):7829-34, 2001;Penfold et al., Proc Natl Acad Sci USA 96(17):9839-9844, 1999; Benedictet al., J Immunol 162(12): 6967-6970, 1999; Spencer et al., J Virol76(3):1285-92, 2002). These sophisticated immune evasion strategiesmight explain CMV's ability to re-infect immune hosts (Plotkin et al., JInfect Dis 159(5): 860-865, 1989; Boppana et al., N Engl J Med344(18):1366-1371, 2001; and see below).

Despite this impressive persistence and immune evasion, and despiteCMV's ability to replicate in a wide variety of cell types, overtdisease in chronic CMV infection is exceedingly rare. Indeed, given itsessentially apathogenic nature in normal children and adults, CMV wouldlikely be an obscure virus, if not for the discovery in the 1950s of itsinvolvement with fetal/neonatal cytomegalic inclusion disease—animportant medical problem because of its associated damage to the CNSand sensory organs—and later, its appearance as one of the most commonand devastating opportunistic pathogens in the settings of organ/bonemarrow transplantation and AIDS. These situations have one strikingcommonality—immunodeficiency, particularly in cell-mediated immunity,related to immunologic immaturity, pharmacologic immunosuppression(transplantation), or the progressive immunodeficiency of HIV infection.This well-characterized relationship between immunity and CMV diseasesuggests that CMV infection exemplifies a complex host-parasiterelationship in which a delicate balance has been evolutionarily‘negotiated’ between viral mechanisms of pathogenesis, persistence, andimmune evasion and the host immune response. In essence, the virus isnot eliminated from the host and has relatively free rein to replicatein excretion sites; yet host immunity restricts replication in mostsites—those in which such replication would lead to disease. With thisbalance, the virus enjoys pervasiveness in a large host population thatcould not occur with unchecked pathogenicity. As for the host, theability of a normal immune system to essentially eliminate pathogenicitywithin reproductively relevant members of the population suggests thatthe support of this pathogen does not pose a significant evolutionaryhandicap.

Although the immunologic requirements for maintaining this host-virusbalance are not yet well characterized, the immunologic ‘resources’specifically devoted to CMV are known to be quite large. The median CD4+T cell response frequencies to human CMV (HCMV) are 5-10 fold higherthan the median CD4+ T cell response frequencies to (whole)non-persistent viruses such as mumps, measles, influenza, andadenovirus, and even other persistent viruses such as herpes simplex andvaricella-zoster viruses. High frequency CD8+ T cell responses toparticular HCMV epitopes or ORFs are also typical. The Applicants havequantified total blood CD4+ and CD8+ T cell responses to the entire CMVgenome (using ˜14,000 overlapping peptides, cytokine flow cytometry, anda large cohort of HLA-disparate CMV-seropositive subjects). TheApplicants have ‘interrogated’ all of these peptides (217 ORF mixes) in24 CMV seropositive and 5 seronegative subjects and have found that whentotaled, the median frequencies of CMV-specific CD4+ and CD8+ T cells inCMV-seropositive subjects are 5-6% for the total CD4+ or CD8+ T cellpopulations (which corresponds to 10-11% of the memory populations)(FIGS. 1, 2). The total responses in seronegative subjects are less than0.5%, a number that represents the ‘background’ of this summationanalysis. Astonishingly, in some individuals, CMV-specific T cellsaccount for more than 25% of the memory T cell repertoire in peripheralblood. This T cell response is broad—each subject recognizes an averageof 19 HCMV ORFs (˜equally distributed among CD4 and CD8 responses), andat least 28 ORFs provoke significant CD4+ or CD8+ T cell responses(≧0.2% of peripheral blood memory population) in 20% or more of thesubjects tested.

The precise mechanisms by which these large CMV-specific T cellpopulations are generated and maintained following infection are notunderstood, but probably relate to the ability of this virus to maintaininfection, including active, productive infection in localmicroenvironments, in the face of a substantial immune response over thelife of the host. In addition, CMV is capable of re-infecting fullyimmune hosts (Boppana et al., N Engl J Med 344(18):1366-1371, 2001;Plotkin et al., J Infect Dis 159(5):860-865, 1989), and it is shownherein in the RM model that experimental re-infection significantlyincreases steady state frequencies of CMV-specific T cells, indicatingthat periodic re-infection plays a central role in the extraordinarybuild-up of CMV-specific T cell response frequencies. Finally, theabilities of HCMV to infect professional Ag-presenting cells(macro-phages, dendritic cells), and produce abundant dense bodies(enveloped tegument protein complexes that can ‘infect’ target cells butlack genetic material) might contribute to the generation of theserobust T cell responses.

Importantly, the characteristics of the antiviral antibody response toHCMV mirror those mentioned above for antiviral T cell immunity.HCMV-specific antibody responses are notable for their reactivity with alarge number of viral proteins and by the persistence of stable titersagainst viral proteins for decades. Antibodies specific for HCMV-encodedproteins, including those against structural proteins made only duringvirus replication, develop rapidly after primary infection and aremaintained at significant titers, likely because of the persistentexpression of virus-encoded proteins. Antibody specific for HCMV is alsopresent on mucosal surfaces, perhaps as a result of the tropism of thisvirus for secretory glands. Interestingly, anti-HCMV antibody responsesalso boost following both community-acquired re-infections andre-infections in transplant recipients.

As indicated above, RMs, the most utilized animal model of lentivirus(SIV) infection, display a natural CMV infection that closely mimicshuman infection with HCMV in terms of epidemiology, patterns ofinfection and disease in immunocompetent and immunodeficient hosts,particularly including RhCMV's role as a major opportunistic pathogen ofSIV-infected monkeys. Not surprisingly, this biologic relatedness isreflected by genetic relatedness. Early work revealed high homologybetween the HCMV genes gB, IE1 and UL121-117 and their RhCMV homologues(Kravitz et al., J Gen Virol 78(Pt 8):2009-2013, 1997; Barry et al.,Virology 215(1):61-72, 1996). The Applicants obtained and analyzed thecomplete sequence of RhCMV strain 68.1, and identified 236 potentialORFs of 100 or more amino acids that are positionally arranged insimilar fashion as their HCMV counterparts. Of these 236 ORFs, 138(58.47%) are clearly homologous to known HCMV proteins. Some of theRhCMV that are homologous to HCMV ORFs known to be nonessential forreplication in fibroblasts are listed in Table 1 below. In someembodiments, these ORFs are sites for insertion of pathogenic antigensfor expression in the disclosed CMV-based vaccine vectors.

In comparison, murine CMV encodes 170 ORFs, of which 78 (45.9%) arehomologous to known HCMV proteins. Importantly, in contrast to murineCMV, RhCMV encodes a full complement of HCMV-like immune evasion genes,and tegument proteins with sufficient homology to HCMV to form densebodies.

TABLE 1 Representative examples of RhCMV ORFs with homologues in theHCMV genome RhCMV ORFs HCMV ORFs RhCMV mutant Rh0l RL1 Rh05 UL153 Rh17UL4 Rh19 UL7 Rh20 UL6 Rh31 UL13 Rh33 UL14 25698, 25739 Rh35a UL19 Rh36UL20 Rh40 UL23 31242 Rh42 UL24 31782, 32619 Rh43 UL25 33323, 33711 Rh54UL31 Rh56 UL33 Rh59 UL35 Rh68 UL42 Rh69 UL43 54274 Rh72 UL45 57859,58210 Rh107 UL78 Rh123 UL88 122332, 122472 Rh143 UL111A Rh148 UL116Rh151/2 UL118/9 Rh155 UL121 155860 Rh158 UL147 Rh159 UL148 165110 Rh160UL132 Rh160a UL130 Rh162 UL145 Rh163 UL144 Rh164 UL141 Rh181 US1 Rh182US2 Rh184 US3 Rh189 US11 Rh190 US12 Rh192 US13 Rh198 US17 Rh199 US18Rh200 US19 Rh201 US20 Rh202 US21 Rh203 US22 Rh221 US29 Rh223 US30 Rh225US31

Each annotated RhCMV ORF, as well as several previously unrecognizedORFs (Rh35a and Rh160a), was compared to the full set of HCMV ORFs usingthe BlastP algorithm. Scores with a significance of ≦10⁻⁵ wereconsidered matches. If more than one RhCMV ORF corresponded to an HCMVORF (e.g., Rh111 and 112 are homologous to UL83) the RhCMV ORF wasexcluded from the list. Nine of the RhCMV ORFs have been mutated byinsertion of a transposon, and insert sites are indicated. The Rh151/2ORFs correspond to the spliced HCMV UL118/9 ORFs.

Basically, BlastP was used to search the RhCMV genome for ORFs that arehomologous to HCMV ORFs known to be nonessential for replication infibroblasts. These nonessential HCMV ORFs were identified: (i) from theliterature; (ii) from transposon mutagenesis of the AD169 strain ofHCMV; and (iii) from the fact that they are in clinical isolates but notthe AD169 laboratory strain. A total of 48 RhCMV ORFs met these criteriaand they are listed in Table 1. The HCMV homologues of three of theseORFs (Rh182, 184 and 189) are known to be immunomodulatory genes, andthree additional immunomodulatory ORFs (Rh185, Rh186 and Rh187) were notidentified in the BlastP analysis because their HCMV homologues weredeleted during the BAC cloning of the clinical HCMVs that weresequenced. To confirm the relationship between the RhCMV and HCMV ORFs,ClustalW was used to perform multiple sequence alignments. Each RhCMVORF was compared to the corresponding ORFs from four clinical isolatesof HCMV. This analysis demonstrated that each RhCMV ORF has an orthologin all four HCMV clinical isolates that have been sequenced, and itconfirmed that the ORFs from the rhesus and human viruses are indeedrelated, ruling out the possibility that the original BlastP scoresresulted from short homologies in otherwise unrelated proteins.

In particular exemplary vectors, two gene blocks are deleted: Rh182-189,which contains homologues to the known HCMV immunomodulatory genesUS2-11, and Rh158-166, which contains genes that are present in clinicalisolates but missing in laboratory strains of HCMV.

The homology between HCMV and RhCMV infections extends to theirrespective immune responses as well. As shown below and in FIGS. 3 and4, the RM T cell response to RhCMV in peripheral blood is similar tothat in the human in both size and the CMV ORFs targeted. Moreover, theincreased accessibility of the RM model has allowed investigation on thefrequencies of these RhCMV-specific T cells in tissue sites, and theimmunologic response to RhCMV re-infection. With regard to the former,as illustrated in FIG. 5, the representation of CMV-specific T cells inthe memory repertoire at the pulmonary tissue:air interface can be trulyenormous, often more than 10-fold higher than in peripheral blood. Withregard to the latter, inoculation of CMV-immune RM with live (but notinactivated) wild-type RhCMV results in a dramatic boosting ofCMV-specific T cell (both CD4+ and CD8+) and antibody responses (FIG.6). The boosted antibody titers appear to return to baseline after about2 months, but importantly, the peripheral blood frequencies ofCMV-specific CD4+ and CD8+ T cells appear to stabilize at levels 50-100%higher than their previous baseline—suggesting periodic re-infections orovert re-activations substantially contribute to the high frequency ofRhCMV-specific T cells observed in most adult RM.

Many of the recognized characteristics of CMV make this virus highlyattractive as a vaccine vector, such as a vaccine vector for aninfectious agent or cancer. First, CMV's capability to elicit strongantibody responses and robust T cell responses focused on mucosal sites(FIGS. 3-5) is of relevance to host defense (particularly for SIV/HIV).It is believed by the Applicants that these strong responses reflect asteady state situation, maintained indefinitely, rather than thepost-boost peak responses that are often highlighted in prior artvaccine studies.

Second, CMV has the ability to re-infect immune hosts, and generate newimmune responses with such re-infections. To confirm this phenomenondirectly with RhCMV, a particular RhCMV vector was constructed thatencodes SIV gag (FIG. 7). This vector showed clear-cut gag expression byboth immunofluorescence and western blot (FIG. 8), and demonstrated invitro growth kinetics indistinguishable from that of wild-type virus.When subcutaneously administered (5×10⁶ PFU) to 4 RhCMV seropositive RM(224 days after primary infection), a similar (clinically asymptomatic)boosting of the RhCMV-specific T cell and antibody response as describedin FIG. 6 was observed. As previously observed in re-infection (FIG. 6),real-time PCR did not identify RhCMV, either wild-type or thegag-recombinant, in blood or lung lavage mononuclear cells at any timepoint following viral inoculation. However, urine from day 127 postre-infection was weakly positive for gag expression by ELISA, suggestingthe presence of the RhCMV-gag vector. These samples were co-cultured toisolate RhCMV, and these in vivo-derived viral preparations wereassessed for gag expression by western blot. As shown in FIG. 8, RhCMVco-cultures from all 4 animals expressed immunoreactive gag,definitively establishing the presence of the administered recombinantvirus in secretory sites. Retrospective analysis of urine at earlierpost-re-infection time points revealed the presence of thegag-expressing RhCMV vector by day 7 in one RM and by day 21 in theother 3. Moreover, the gag-expressing RhCMV vector has also beendetected in saliva, and remains present in urine at least through day237 post re-infection.

These data have two critical implications. First, they unequivocallydemonstrate that CMV is capable of re-infecting immune subjects,effectively competing with pre-existent wild-type virus, and somehowfinding its way to its usual ‘ecological’ niche despite strongly boostedcellular and humoral immunity directed at CMV. Second, they demonstratethe in vivo stability of RhCMV vectors expressing exogenous neoAgs,indicating that these vectors are able to persistently infect inoculatedsubjects, and indefinitely maintain expression of the inserted,exogenous antigen-encoding genes.

This RhCMV-gag re-infected cohort was also used to assess the ability ofRhCMV vectors to initiate a de novo immune response in the face of themassive CMV-specific memory boost associated with re-infection.

As shown in FIGS. 9 and 10, all inoculated RM developed gag-specificCD4+ and CD8+ T cell responses in blood, as early as day 7 postre-infection. These blood responses peaked in the first month followingre-infection at 0.2%-0.6% of memory cells, declining thereafter to astable plateau in the 0.1%-0.2% range. SIV gag-specific antibodies werealso induced by day 14 post-infection, increasing through day 91post-infection, prior to achieving a stable plateau (FIG. 12).Re-administration of the same RhCMV-gag vector (at day 238 postre-infection) dramatically boosted both examined, these responsesremained higher than the previous ‘set points,’ suggesting establishmentof higher plateau levels. Significantly, as previously shown forRhCMV-specific T cell responses, blood frequencies of these CMV-vectoredgag-specific T cell responses substantially (>10×) underestimated thefrequency of SW-specific T cells in a tissue effector site—the lung(FIGS. 10 and 11). Gag-specific CD4+ and CD8+T cells were found in lunglavage fluid by day 7 post the initial RhCMV(gag) re-infection in allRM, peaking with frequencies as high as 10% (day 42-56 postre-infection) before achieving stable plateau levels in the 1%-3% range.Boosting (second re-infection) resulted in a sharp spike in thesefrequencies with return to the previous plateau level in 2 RM, and whatappear to be (at day 70 post re-infection #2) slightly higher plateaulevels in the other 2 animals. Significantly, the frequency anddistribution of gag-specific T cells in these RhCMV(gag)-immunized RMare comparable to what was observed in RM ‘immunized’ with attenuatedSIVmac239(Δnef) that effectively controlled I. V. challenge withwild-type SIVmac239 (FIG. 13). It should also be noted that in all ofthe RMs studied, the observed gag-specific responses occurred in thesetting of significant boosting of the RhCMV-specific responses (FIG.9); indeed, it is possible these recall CMV-specific responses acted asan adjuvant for the new gag-specific response. Significantly, these datademonstrate the ability of RhCMV to function effectively as a vector forneoAgs in RhCMV-immune RM.

According to particular aspects of the present disclosure, the uniqueability of CMV to effectively ‘vector’ neoAg responses in CMV-immunesubjects has several highly significant implications for their utilityin a vaccine. First, by virtue of the fact that any healthy CMV+subjects have operationally demonstrated their ability to controlwild-type CMV infection, their risk of morbidity after administration ofCMV vectors, even vectors based on an otherwise unmodified CMV genome,would be expected to be minimal. Although fetuses of CMV+ mothers can insome circumstances be infected, this risk can be averted by simply notproviding CMV-based vaccines to pregnant females. Incorporation ofsafety (e.g., inducible suicide) mechanisms and/or strategic genedeletion (so as to reduce pathogenic potential without sacrificingimmunogenicity and persistence) would be expected to reduce thepossibility of vaccine morbidity even further. It should also be notedthat other well-studied herpesviruses that could potentially be used aspersistent and perhaps re-infection capable vectors have one or morefeatures that mitigate against such use. For example, theγ-herpesviruses Epstein Barr Virus (EBV) and Kaposi's Sarcoma HerpesVirus (KSHV) are firmly associated with malignancies, whereas CMV isnot. Additionally, α herpesviruses (herpes simplex) lack the generalizedT cell immunogenicity of CMV and because of neurovirulence (e.g., herpesencephalitis) would appear to have considerable more pathogenicpotential in otherwise immunocompetent individuals.

The second implication of CMV's re-infection capability and thedemonstration herein that a subsequent inoculation with the same RhCMVvector elicits a strong immunologic boost to the recombinant (SIV) geneproduct (FIG. 11) is that, unlike essentially all other viral vectorscurrently in use or in development, CMV vectors can likely be usedrepeatedly. Indeed, according to particular embodiments of the presentdisclosure, individuals are serially vaccinated with different CMVvectors so as to generate responses to new epitopes.

In one embodiment herein, it is disclosed that a CMV-driven,anti-HIV/SIV immune response (e.g., in place at the time of HIV/SIVexposure) is sufficient to contain viral replication, blunt the initialviral diaspora, prevent immunopathogenicity, and establish anon-progressive infection. Aspects of the present disclosure co-optCMV's eons of evolution, and provide strategically engineered andoptimized CMV as a heterologous ORF-encoding vaccine vector. CMV'scombination of 1) remarkable immunogenicity, 2) low pathogenicity, 3)persistence, 4) widespread tissue dissemination, and 5) the ability tore-infect CMV+ hosts is substantially and fundamentally different fromother vaccine vectors in development. Indeed, the issue of persistence,by itself, makes this approach substantially worthy. According toparticular aspects, long-term antigen exposure, as is possible with thedisclosed CMV-based vaccine vectors, correlates with a qualitativelydifferent and functionally superior anti-lentiviral immune response.

For safety considerations, CMV vectors, with all their unique potential,must be handled appropriately. Therefore, in some embodiments, theCMV-based vaccination vectors are used to elicit neoAg immunity in CMV+hosts, who have—by their healthy, CMV+ status—established their abilityto contain this virus. Although no live virus vector is without risk, inthe appropriate setting (such as in the context of demonstrablyimmunocompetent, CMV+ pre-adolescents), the risk of serious CMV vectorpathogenicity, even with non-safety modified vectors, should be minimal.According to further aspects of the present disclosure, even therelatively low disease-inducing potential of wild-type CMV isabrogatable by genetic manipulation of the CMV vector withoutsacrificing immunogenicity or persistence.

According to aspects of the present disclosure, CMV vectors, alone or incombination with other modalities, provide qualitatively superiorcellular and humoral immune responses against infectious disease, suchthat such challenges are significantly contained. Additional aspectsprovide safe vectors without sacrificing the unique persistence andre-infection capabilities. According to yet further aspects, various CMVgenes are deletable without sacrificing vector function, and particularexemplary vectors use the genomic ‘space’ created by such deletions forinsertion of safety constructs (e.g., inducible suicide mechanisms) aswell as larger (poly-cistronic) gene encoding cassettes. This genedeletion approach is designed to retain the balance betweenimmunogenicity/persistence and pathogenicity, and exploits theredundancy of CMV's adaptations in providing engineered RhCMV optimizedvectors.

In some embodiments, RhCMV vectors are designed to reflect genes andbiology that are homologous to HCMV, and optimized RhCMV vector designsare directly applicable to construction of HCMV vector embodiments.

In some embodiments, the CMV vectors disclosed herein encode aheterologous antigen. The heterologous antigen is an antigenicpolypeptide encoded by a heterologous polynucleotide that isincorporated into the recombinant CMV vector. In particular examples,the antigenic polypeptide is derived from a bacterium, fungus,protozoan, virus or tumor. The polypeptide can be anything that isbeneficially used as an antigen to stimulate an immune response, thoughit is contemplated that the benefit of provoking a long-term immuneresponse against a particular antigen will influence its selection.

The antigenic polypeptide sequence may be any length sufficient toelicit the immune response. In particular examples, the polypeptide isat least 8, at least 10, at least 20, at least 30, at least 40, at least50 amino acids long or greater. Likewise, the sequence identity of theantigenic polypeptide need not be identical to the sequence identity tothe native polypeptide in order to be sufficient to maintain specificityof the immune response against the bacterium, protozoan, fungus, virusor tumor. One of skill in art will recognize that the sequence of apolypeptide may be significantly altered while maintaining its antigenicspecificity—that is, the ability to stimulate an antigenic response thatwill still provide responsiveness to the native protein. Thus, inparticular examples, the antigenic polypeptide is at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least98% identical to the polypeptide from which it was derived. Inparticular examples, the polypeptide is an analog of the hostpolypeptide that is found in a different species (xenogeneic).

One of ordinary skill will recognize that the lists of exemplaryheterologous polypeptides discussed herein are neither exhaustive norintended to be limiting. Thus, it will be recognized that the methodsprovided herein are useful for expression of, and thusimmune-stimulation related to, any polypeptide against which it would bebeneficial to generate immunity.

In some embodiments, the heterologous antigen is a polypeptide derivedfrom a pathogenic organism such bacteria, fungi, protozoa, or virus.

Examples of pathogenic viruses include, but are not limited to those inthe following virus families: Retroviridae (for example, humanimmunodeficiency virus (HIV), human T-cell leukemia viruses;Picornaviridae (for example, polio virus, hepatitis A virus, hepatitis Cvirus, enteroviruses, human coxsackie viruses, rhinoviruses,echoviruses, foot-and-mouth disease virus); Caliciviridae (such asstrains that cause gastroenteritis, including Norwalk virus);Togaviridae (for example, alphaviruses (including chikungunya virus,equine encephalitis viruses, Simliki Forest virus, Sindbis virus, RossRiver virus), rubella viruses); Flaviridae (for example, dengue viruses,yellow fever viruses, West Nile virus, St. Louis encephalitis virus,Japanese encephalitis virus, Powassan virus and other encephalitisviruses); Coronaviridae (for example, coronaviruses, severe acuterespiratory syndrome (SARS) virus; Rhabdoviridae (for example, vesicularstomatitis viruses, rabies viruses); Filoviridae (for example, Ebolavirus, Marburg virus); Paramyxoviridae (for example, parainfluenzaviruses, mumps virus, measles virus, respiratory syncytial virus);Orthomyxoviridae (for example, influenza viruses); Bunyaviridae (forexample, Hantaan viruses, Sin Nombre virus, Rift Valley fever virus,bunya viruses, phleboviruses and Nairo viruses); Arenaviridae (such asLassa fever virus and other hemorrhagic fever viruses, Machupo virus,Junin virus); Reoviridae (e.g., reoviruses, orbiviurses, rotaviruses);Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses,BK-virus); Adenoviridae (adenoviruses); Herpesviridae (herpes simplexvirus (HSV)-1 and HSV-2; cytomegalovirus; Epstein-Barr virus; varicellazoster virus; and other herpes viruses, including HSV-6); Poxyiridae(variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (suchas African swine fever virus); Astroviridae; and unclassified viruses(for example, the etiological agents of spongiform encephalopathies, theagent of delta hepatitis (thought to be a defective satellite ofhepatitis B virus).

Examples of bacterial pathogens include, but are not limited to:Helicobacter pylori, Escherichia coli, Vibrio cholerae, Boreliaburgdorferi, Legionella pneumophilia, Mycobacteria sps (such as. M.tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae),Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis,Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus),Streptococcus agalactiae (Group B Streptococcus), Streptococcus(viridans group), Streptococcus faecalis, Streptococcus bovis,Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenicCampylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillusanthracis, corynebacterium diphtheriae, corynebacterium sp.,Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridiumtetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturellamultocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillusmoniliformis, Treponema pallidium, Treponema pertenue, Leptospira,Bordetella pertussis, Shigella flexnerii, Shigella dysenteriae andActinomyces israelli.

Examples of fungal pathogens include, but are not limited to:Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis,Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.

Other pathogens (such as parasitic pathogens) include, but are notlimited to: Plasmodium falciparum, Plasmodium vivax, Trypanosoma cruziand Toxoplasma gondii.

Immunogenic proteins encoded by pathogenic microorganisms are well knownand/or can be determined by those of skill in the art. Thus, thespecific antigen encoded by the recombinant CMV vector can be anyprotein or fragment thereof (such as an epitope or antigenic fragmentthereof) that is specifically expressed by the pathogen and is capableof eliciting an immune response in a subject.

In some embodiments, the recombinant CMV vector encodes an antigen froma virus. The viruses may include but are not limited to Influenza virus,West nile virus, Poliovirus, SIV, HIV, HCMV, Kaposi's sarcoma associatedherpesvirus, Ebola virus, Chikungunya virus, Dengue virus, Monkeypoxvirus, varcella zoster virus, Epstein-barr virus, Herpes simplex 1 virusand Herpes simplex 2 virus. In particular embodiments disclosed herein,the recombinant CMV vector encodes an antigen from influenza virus. Insome examples, the influenza virus antigen is hemagglutinin (HA) orneuraminidase, or an epitope or antigenic fragment thereof. In otherembodiments, the recombinant CMV vector encodes an antigen frommonkeypox virus. In some examples, the monkeypox virus antigen is A35R,or an epitope or antigenic fragment thereof. In other embodiments, therecombinant CMV vector encodes an antigen from West Nile virus. In someexamples, the West Nile virus antigen is capsid (C), membrane (prM/M),envelope (E), NS1, NS2A, NS2B, NS3, NS4A, NS4B or NS5, or an epitope orantigenic fragment thereof. In other embodiments, the recombinant CMVvector encodes an antigen from Chikungunya virus. In some examples, theChikungunya virus antigen is capsid (C), envelope glycoprotein 1 (E1),envelope glycoprotein 2 (E2), envelope glycoprotein 3 (E3) ornon-structural protein 1 (NSP1), or an epitope or antigenic fragmentthereof. In other embodiments, the recombinant CMV vector encodes anantigen from Ebola virus. In some examples, the Ebola virus antigen isnucleoprotein (NP), or an epitope or antigenic fragment thereof. Inother embodiments, the recombinant CMV vector encodes an antigen fromHCV. In some examples, the HCV antigen is core, E1, E2, NS2, NS3, NS4 orNS5, or an epitope or antigenic fragment thereof. In other embodiments,the recombinant CMV vector encodes an antigen from poliovirus. In someexamples, the poliovirus antigen is VP1, or an epitope or antigenicfragment thereof. In other embodiments, the recombinant CMV vectorencodes an antigen from a dengue virus, such as a dengue serotype 1, 2,3, or 4 virus. In some examples, the dengue virus antigen is capsid (C),membrane (prM/M), envelope (E), NS1, NS2A, NS2B, NS3, NS4A, NS4B or NS5,or an epitope or antigenic fragment thereof.

In some embodiments, the recombinant CMV vector encodes a bacterialantigen. In particular embodiments, the antigen is from Clostridiumtetani, the causative agent of tetanus. In some examples, the antigenfrom Clostridium tetani is tetanusC, or an epitope or antigenic fragmentthereof. TetanusC is a non-toxic fragment of tetanus toxin that iscapable of eliciting a protective immune response to tetanus toxin.

In some embodiments, the recombinant CMV vector encodes a fungalantigen.

In some embodiments, the recombinant CMV vector encodes a protozoanantigen. In particular embodiments, the protozoan antigen is an antigenfrom Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale orPlasmodium malariae, each of which can cause malaria (with Plasmodiumfalciparum being the most common causative agent for malaria). In someexamples, the Plasmodium antigen is a pre-erythrocytic antigen, such asCSP or SSP2, or an epitope or antigenic fragment thereof. In otherexamples, the Plasmodium antigen is an erythrocytic antigen, such asAMA1 or MSP1, or an epitope or antigenic fragment thereof.

In other embodiments, the heterologous polynucleotide carried by therecombinant CMV vector encodes a polypeptide derived from a tumor. Thetumor may be the result of any type of cellular proliferative disease orcondition, and may be benign or malignant. In particular examples, thetumor is cancerous. Such tumors can be of any type of cancer including,but not limited to: leukemias, including acute leukemias (such as acutelymphocytic leukemia, acute myelocytic leukemia, acute myelogenousleukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic anderythroleukemia), chronic leukemias (such as chronic myelogenousleukemia, and chronic lymphocytic leukemia), myelodysplastic syndrome,and myelodysplasia, polycythemia vera, lymphoma, (such as Hodgkin'sdisease, all forms of non-Hodgkin's lymphoma), multiple myeloma,Waldenstrom's macroglobulinemia, and heavy chain disease. Examples ofsolid tumors, such as sarcomas and carcinomas, include, but are notlimited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing'stumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreaticcancer, breast cancer, lung cancer, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testiculartumor, bladder carcinoma, melanoma, and CNS tumors (such as a glioma,astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,meningioma, neuroblastoma and retinoblastoma).

Antigenic tumor-derived polypeptides that may be encoded by theheterologous polynucleotide of the described compositions and methodsencompass polypeptides also known as tumor associated antigens (TAAs),and peptides derived therefrom. Many TAAs have been identified. Theseinclude, but are not limited to: human telomerase reverse transcriptase(hTERT), survivin, MAGE-1, MAGE-3, human chorionic gonadotropin,carcinoembryonic antigen, alpha fetoprotein, pancreatic oncofetalantigen, MUC-1, CA 125, CA 15-3, CA 19-9, CA 549, CA 195,prostate-specific membrane antigen, Her2/neu, gp-100, trp-2, mutantK-ras proteins, mutant p53, truncated epidermal growth factor receptor,chimeric protein ^(p210)BCR-ABL; E7 protein of human papilloma virus,EBNA3 protein of Epstein-Barr virus, carcinoembryonic antigen (CEA),β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactiveAFP, thyroglobulin, RAGE-1, MN-CA IX, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA),PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, telomerase,prostate-carcinoma tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase,ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptorand mesothelin (some of these TAAs as well as others are described inNovellino et al., Cancer Immunology and Immunotherapy, 54:187-207,2005).

A list of exemplary tumor antigens and their associated tumors are shownbelow in Table 2.

TABLE 2 Exemplary tumors and their tumor antigens Tumor Tumor AssociatedAntigens Acute myelogenous leukemia Wilms tumor 1 (WT1), preferentiallyexpressed antigen of melanoma (PRAME), PR1, proteinase 3, elastase,cathepsin G Chronic myelogenous leukemia WT1, PRAME, PR1, proteinase 3,elastase, cathepsin G Myelodysplastic syndrome WT1, PRAME, PR1,proteinase 3, elastase, cathepsin G Acute lymphoblastic leukemia PRAMEChronic lymphocytic leukemia Survivin Non-Hodgkin's lymphoma SurvivinMultiple myeloma NY-ESO-1 Malignant melanoma MAGE, MART, Tyrosinase,PRAME GP100 Breast cancer WT1, herceptin, epithelial tumor antigen (ETA)Lung cancer WT1 Ovarian cancer CA-125 Prostate cancer PSA Pancreaticcancer CA19-9, RCAS1 Colon cancer CEA Renal cell carcinoma (RCC)Fibroblast growth factor 5 Germ cell tumors AFP

Successful tumor immunotherapy is hampered by the poor immunogenicity oftumor epitopes and the fact that the immune system often responds tothem by immune tolerance rather than by initiating a potent effectorresponse. An ideal immunotherapeutic tumor vaccine would elicit animmune response that is (a) robust, (b) effective, (c) resistant to thedevelopment of immune tolerance, (d) long-lived, and (e) after initialtumor control, effective at the task of immune surveillance againstrecurrences and metastases. The robust, life-long immune responseelicited by CMV infection, and the ability of CMV to overcomeself-tolerance, suggest that CMV may be an ideal vaccine vector fortumor immunotherapy.

Recombinant CMV vectors encoding a tumor antigen, and compositionsthereof, may be administered singly, or administered in combination withone or more pharmaceutically active compounds. For example, thecompounds may be co-administered with other anti-cancer compounds suchas alkylating agents, antimetabolites, natural products, hormones andtheir antagonists, other miscellaneous agents, or any combination ofthese. Additional anti-cancer agents can be administered prior to, atthe same time, or following administration of the recombinant CMVvector.

Examples of alkylating agents include nitrogen mustards (such asmechlorethamine, cyclophosphamide, melphalan, uracil mustard orchlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (suchas carmustine, lomustine, semustine, streptozocin, or dacarbazine).Examples of antimetabolites include folic acid analogs (such asmethotrexate), pyrimidine analogs (such as 5-FU or cytarabine), andpurine analogs, such as mercaptopurine or thioguanine Examples ofnatural products include vinca alkaloids (such as vinblastine,vincristine, or vindesine), epipodophyllotoxins (such as etoposide orteniposide), antibiotics (such as dactinomycin, daunorubicin,doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (suchas L-asparaginase). Examples of miscellaneous agents include platinumcoordination complexes (such as cis-diamine-dichloroplatinum II alsoknown as cisplatin), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (such as procarbazine), and adrenocorticalsuppressants (such as mitotane and aminoglutethimide).

Examples of hormones and antagonists include adrenocorticosteroids (suchas prednisone), progestins (such as hydroxyprogesterone caproate,medroxyprogesterone acetate, and magestrol acetate), estrogens (such asdiethylstilbestrol and ethinyl estradiol), antiestrogens (such astamoxifen), and androgens (such as testerone proprionate andfluoxymesterone). Examples of the most commonly used chemotherapy drugsinclude Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU,Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU,Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin,Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, suchas docetaxel), Velban, Vincristine, VP-16, while some more newer drugsinclude Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11),Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin),Xeloda (Capecitabine), Zevelin and calcitriol. Non-limiting examples ofimmunomodulators that can be used include AS-101 (Wyeth-Ayerst Labs.),bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocytemacrophage colony stimulating factor; Genetics Institute), IL-2 (Cetusor Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG(from Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosisfactor; Genentech).

For the treatment of cancer, administration of a recombinant CMV vectorencoding a tumor antigen may be preceded by, or followed by, one or moreadditional therapies to treat the cancer, such as surgical resection ofthe tumor or radiation therapy.

Also disclosed herein are recombinant CMV vectors, such as RhCMV andHCMV vectors, having a deletion in one or more genes that are essentialfor or augment CMV replication, dissemination or spreading. Thus, thesevectors are referred to as “replication-deficient” CMV vectors. As usedherein, “replication-deficient” encompasses CMV vectors that are unableto undergo any replication in a host cell, or have a significantlyreduced ability to undergo viral replication. In some examples, thereplication-deficient CMV vectors are able to replicate, but are unableto disseminate since they are incapable of infection neighboring cells.In some examples, the replication-deficient CMV vectors are able toreplicate, but are unable to spread since they are not secreted frominfected hosts.

CMV essential and augmenting genes are well known in the art (see, forexample, Dunn et al., Proc. Natl. Acad. Sci. USA 100(24):14223-14228,2003; and Dong et al., Proc. Natl. Acad. Sci. USA 100(21):12396-12401,2003), and are described herein. In some embodiments, the recombinantRhCMV or HCMV vector includes a deletion in one gene that is essentialfor or augments virus replication, dissemination or spreading. In otherembodiments, the recombinant RhCMV or HCMV vector includes a deletion inmultiple (such as, but not limited to, two, three or four) genesessential for or augmenting CMV replication, dissemination or spreading.The deletion need not be a deletion of the entire open reading frame ofthe gene, but includes any deletion that eliminates expression offunctional protein.

In some embodiments, the recombinant RhCMV or HCMV vector includes adeletion in one or more genes selected from UL82 (encoding pp71) (seeFIGS. 36-38), UL94 (encoding the UL94 protein), UL32 (encoding pp150),UL99 (encoding pp28), UL115 (encoding gL) and UL44 (encoding p52), or ahomolog thereof (i.e., the RhCMV homolog of these HCMV genes).

Replication-deficient RhCMV and HCMV vectors disclosed herein caninclude a nucleic acid sequence encoding a heterologous antigen, such asa pathogen-specific antigen or a tumor antigen. As disclosed for otherrecombinant RhCMV and HCMV vectors described herein,replication-deficient RhCMV and HCMV vectors can be used to elicit animmune response in a subject against the encoded heterologous antigen.

A recombinant RhCMV vector having a deletion in gene UL82 (which encodesthe pp71 protein) is severely impaired in its ability grow in vitro andto spread in vivo, but still elicits a robust T cell immune responseagainst CMV. Thus, it is contemplated herein to use such areplication-deficient vector as a vaccine against CMV itself (see FIGS.36-38)

In advantageous embodiments of the present invention, a recombinant CMVvector, such as a RhCMV or a HCMV vector may have deletions in generegions non-essential for growth in vivo. Such gene regions include, butare not limited to, the RL11 family, the pp65 family, the US12 familyand the US28 family. In particular, RhCMV gene regions that may bedeleted include gene regions Rh13-Rh29, Rh111-RH112, Rh191-Rh202 andRh214-Rh220. More particularly, the RhCMV gene regions that may bedeleted include Rh13.1, Rh19, Rh20, Rh23, Rh24, Rh112, Rh190, Rh192,Rh196, Rh198, Rh199, Rh200, Rh201, Rh202 and Rh220. In anotherembodiment, HCMV gene regions that may be deleted include gene regionsRL11 (L), UL6, UL7 (L), UL9 (L), UL11 (E), UL83 (L) (pp65), US12 (E),US13 (E), US14 (E), US17 (E), US18 (E), US19 (E), US20 (E), US21 andUL28.

Recombinant CMV vectors, or compositions thereof, can be administered toa subject by any of the routes normally used for introducing recombinantvirus or viral vectors into a subject. Methods of administrationinclude, but are not limited to, intradermal, intramuscular,intraperitoneal, parenteral, intravenous, subcutaneous, vaginal, rectal,intranasal, inhalation or oral. Parenteral administration, such assubcutaneous, intravenous or intramuscular administration, is generallyachieved by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution or suspension in liquid prior to injection, or asemulsions. Injection solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.Administration can be systemic or local.

Compositions including recombinant CMV vectors are administered in anysuitable manner, such as with pharmaceutically acceptable carriers.Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent disclosure.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Some of the compositions may potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

Administration can be accomplished by single or multiple doses. The doseadministered to a subject in the context of the present disclosureshould be sufficient to induce a beneficial therapeutic response in asubject over time. The dose required will vary from subject to subjectdepending on the species, age, weight and general condition of thesubject, the severity of the infection being treated, the particularcomposition being used and its mode of administration. An appropriatedose can be determined by one of ordinary skill in the art using onlyroutine experimentation.

Provided herein are pharmaceutical compositions which include atherapeutically effective amount of the recombinant CMV vector alone orin combination with a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers include, but are not limited to,saline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. The carrier and composition can be sterile, andthe formulation suits the mode of administration. The composition canalso contain minor amounts of wetting or emulsifying agents, or pHbuffering agents. The composition can be a liquid solution, suspension,emulsion, tablet, pill, capsule, sustained release formulation, orpowder. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oralformulations can include standard carriers such as pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, and magnesium carbonate. Any of the common pharmaceuticalcarriers, such as sterile saline solution or sesame oil, can be used.The medium can also contain conventional pharmaceutical adjunctmaterials such as, for example, pharmaceutically acceptable salts toadjust the osmotic pressure, buffers, preservatives and the like. Othermedia that can be used with the compositions and methods provided hereinare normal saline and sesame oil.

The recombinant CMV vectors described herein can be administered aloneor in combination with other therapeutic agents to enhance antigenicity.For example, the CMV vectors can be administered with an adjuvant, suchas Freund's incomplete adjuvant or Freund's complete adjuvant.

Optionally, one or more cytokines, such as IL-2, IL-6, IL-12, RANTES,GM-CSF, TNF-α, or IFN-γ, one or more growth factors, such as GM-CSF orG-CSF; one or more molecules such as OX-40L or 41 BBL, or combinationsof these molecules, can be used as biological adjuvants (see, forexample, Salgaller et al., 1998, J. Surg. Oncol. 68(2):122-38; Lotze etal., 2000, Cancer J. Sci. Am. 6(Suppl 1):S61-6; Cao et al., 1998, StemCells 16(Suppl 1):251-60; Kuiper et al., 2000, Adv. Exp. Med. Biol.465:381-90). These molecules can be administered systemically (orlocally) to the host.

A number of means for inducing cellular responses, both in vitro and invivo, are known. Lipids have been identified as agents capable ofassisting in priming CTL in vivo against various antigens. In certainembodiments, the use of liposomes, nanocapsules, microparticles,microspheres, lipid particles, vesicles, and the like, are contemplatedfor the introduction of the compositions of the present disclosure intosuitable host cells. In particular, the compositions of the presentdisclosure may be formulated for delivery either encapsulated in a lipidparticle, a liposome, a vesicle, a nanosphere, or a nanoparticle or thelike.

Such formulations may be preferred for the introduction ofpharmaceutically-acceptable formulations of the nucleic acids orconstructs disclosed herein. The formation and use of liposomes isgenerally known to those of skill in the art. Liposomes have beendeveloped with improved serum stability and circulation half-times (U.S.Pat. No. 5,741,516). Further, various methods of liposome and liposomelike preparations as potential drug carriers have been reviewed (U.S.Pat. No. 5,567,434; U.S. Pat. No. 5,552,157; U.S. Pat. No. 5,565,213;U.S. Pat. No. 5,738,868 and U.S. Pat. No. 5,795,587).

Liposomes have been used successfully with a number of cell types thatare normally resistant to transfection by other procedures including Tcell suspensions, primary hepatocyte cultures and PC 12 cells. Inaddition, liposomes are free of the DNA length constraints that aretypical of viral-based delivery systems. Liposomes have been usedeffectively to introduce genes, drugs, radiotherapeutic agents, enzymes,viruses, transcription factors and allosteric effectors into a varietyof cultured cell lines and animals. In addition, several successfulclinical trials examining the effectiveness of liposome-mediated drugdelivery have been completed. Furthermore, several studies suggest thatthe use of liposomes is not associated with autoimmune responses,toxicity or gonadal localization after systemic delivery.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

Liposomes bear resemblance to cellular membranes and are contemplatedfor use in connection with the present disclosure as carriers for theCMV vector compositions. They are widely suitable as both water- andlipid-soluble substances can be entrapped, i.e., in the aqueous spacesand within the bilayer itself, respectively. It is possible that thedrug-bearing liposomes may even be employed for site-specific deliveryof active agents by selectively modifying the liposomal formulation.

Some embodiments of the invention relate to the alteration of RhCMV/SIVvector tropism to prevent replication in cells and tissues associatedwith viral spread and pathogenesis. The tropism-defective vector eitherlacks genes required for optimal growth in certain cell types or thevector is modified to contain targets for tissue-specific micro-RNAs ingenes that are essential for viral replication.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

EXAMPLES Example 1 Recombinant HCMV and RhCMV Vectors

This example describes exemplary recombinant HCMV and RhCMV vectorsencoding heterologous antigens, such as pathogen-specific antigens orcancer antigens. Particular aspects of the disclosure providerecombinant RhCMV and HCMV vectors that are deficient or impaired intheir ability to replicate in vitro and in vivo, disseminate within thehost, or spread from host to host. Other aspects provide RhCMV and HCMVvectors that can be growth-modulated in vivo (e.g., by oraladministration of the antibiotic doxycycline). Heterologous antigenexpression may be under the control of promoters of different kineticclasses with respect to the CMV infection cycle (e.g.,EF1α—constitutive; MIE—immediate early; pp65—early; gH—late).

In particular embodiments, RhCMV and HCMV vectors lack immune modulatorygenes (e.g., Rh158-166 and Rh182-189 (US2-11)) to enhance vectorimmunogenicity, safety and heterologous gene carrying capacity of thevector. For example, HCMV encodes at least four different gene products,gpUS2, gpUS3, gpUS6 and gpUS11 that interfere with antigen presentationby MHC I (37). All four HCMV MHC evasion molecules are encoded in theunique short region of HCMV and belong to the related US6 gene family.Additional HCMV immunomodulators include, but are not limited to UL118,UL119, UL36, UL37, UL111a, UL146, UL147, etc. Likewise, RhCMV containshomologous and analogous immune modulatory genes that can be deleted ormodified to enhance vector immunogenicity, safety and heterologous genecarrying capacity of the disclosed vaccine vectors.

In additional embodiments, recombinant RhCMV and HCMV vectors arefurther optimized for anti-pathogen or anti-tumor immunogenicity byinsertion of multiple antigen genes. Alternatively, several vectors,each having a single inserted antigen may be used for co-administration.

In additional embodiments, recombinant RhCMV and HCMV vectors containLoxP sites (e.g., RhCMV/AntigenLoxPCre) strategically placed in the CMVgenome to flank an essential region of the viral genome, in combinationwith a tetracycline (Tet)-regulated Cre recombinase. Followingimmunization, doxycycline (Dox)-mediated induction of Cre recombinaseenables in vivo inactivation of RhCMV/AntigenLoxPCre by cleavage at theLoxP sites.

In additional embodiments, the recombinant RhCMV or HCMV vector includesa deletion in one or more genes that are essential for or augment virusreplication inside the infected cell, dissemination within the host, andspreading between hosts. Specific examples of essential or augmentinggenes include, but are not limited to, UL82, UL94, UL32, UL99, UL115 orUL44, or a homolog thereof. Other essential genes are known in the artand are contemplated for deletion in the disclosed RhCMV and HCMVvectors (see, for example, Dunn et al., Proc. Natl. Acad. Sci. USA100(24):14223-14228, 2003; Dong et al., Proc. Natl. Acad. Sci. USA100(21):12396-12401, 2003).

Construction and Characterization of the RhCMV BAC

The development of BAC technology to clone large segments of genomic DNAcoupled with sophisticated λ phage-based mutagenesis systems hasrevolutionized the field of herpes virology enabling genetic approachesto analyze the virus. This system was used, for example, to construct anRhCMV BAC (RhCMV BAC-Cre) containing the complete RhCMV strain 68-1genome. The RhCMV BAC-Cre was derived from an infectious, pathogenicRhCMV 68-1/EGFP recombinant virus (16). RhCMV BAC-Cre contains a BACcassette inserted at a single LoxP site within the Rh181 (US1)/Rh182(US2) intergenic region of RhCMVvLoxP. Insertion of the BAC cassette atthis site results in the generation of LoxP sequences flanking thecassette. As the BAC cassette contains a Cre gene that is expressed ineukaryotic cells, transfection of this ‘self-excising’ RhCMV BAC-Creinto fibroblasts results in efficient excision of the BAC cassette,reconstituting virus (designated RhCMVvLoxP). Characterization of thegrowth of the BAC-reconstituted virus (RhCMVvLoxP) in vitro and in vivodemonstrates that the various genetic manipulations did not alter the WTproperties of the virus. The genomic structure of RhCMVvLoxP isidentical to that of WT RhCMV except for the residual LoxP site. Thepresence of the LoxP sequence does not alter the expression profiles ofneighboring Rh181 (US1) and Rh182 (US2) or distal (IE2) genes.RhCMVvLoxP replicates with WT kinetics both in tissue culture and inRhCMV seronegative immunocompetent RMs (n=2). Analysis of tissues fromone animal terminated at 6 months post-inoculation demonstrated thepresence of both RhCMV DNA and IE1-expressing cells in the spleen,consistent with the persistent gene expression observed in previousstudies with WT virus. Both RMs developed vigorous anti-RhCMV antibodytiters comparable to those observed in naturally infected animals. Takentogether, these observations demonstrate that RhCMVvLoxP isphenotypically WT and is suitable to construct site-specific alterationsfor the development of vaccine vectors.

Exemplary HCMV and RhCMV Vaccine Vectors

FIG. 15 schematically depicts construction of RhCMV and HCMV vaccinevectors according to particular aspects of the present disclosure.Heterologous pathogen antigen(s) are inserted into RhCMV or HCMVbacterial artificial chromosomes (BACs) by E/T and Flp-mediatedrecombination. The schematic shows, for example, a generalized strategyfor insertion of an epitope-tagged pathogen antigen into the non-codingregion between rh213 and Rh214 of RhCMV. This strategy can be similarlyused for insertion of heterologous pathogen antigens at other definedsites within the RhCMV/HCMV genome, as well as insertion of multipleantigens at single or multiple sites within the genome. The geneencoding the epitope-tagged pathogen antigen is inserted into the BACgenome using E/T recombination. Following selection of recombinant BACson the basis of antibiotic resistance (such as Kan), the resistance geneis removed by Flp-mediated recombination. Recombinant RhCMV and HCMVvaccine vectors are reconstituted by transfection of recombinant BACsinto RhCMV/HCMV permissive cells (AgX, pathogen or cancer antigen; Tag,epitope tag; pA, polyadenylation site; Kan, kanamycin resistance genefor selection in bacteria).

FIG. 16 shows an exemplary tetracycline-regulated RhCMV/HCMV ‘safety’vaccine vector. This RhCMV/HCMV safety vector contains two interactivegenetic components within the RhCMV/HCMV genome that together enableTet-induced vector inactivation. A Tet-inducible Cre recombinase gene(Cre) inserted within the viral genome enables induction of Crerecombinase expression by treatment with the Tet homologue, doxycycline(Dox) (D). This Tet-regulated system is comprised of a Tet-sensitivereverse-transactivator (rtTA2^(s)-M2) (TA2), a Tet-transrepressor(tTS-kid) (TS) and a tetO₇-CMV minimal promoter unit (white arrow)driving Cre-recombinase expression. The second component necessary forTet-induced inactivation is a pair of LoxP sites located within theviral genome to flank a region of the genome essential for virusreplication (in this case, Rh52-Rh156). In the absence of Dox, thebinding of tTS-kid and lack of binding of rtTA2^(s)-M2 to the tetO₇-CMVminimal promoter unit prevents Cre-recombinase expression. In theabsence of Cre recombinase, the integrity of the RhCMV/HCMV genome ismaintained and the virus replicates normally. Addition of Dox results inthe allosteric modulation of tTS-kid and rtTA2^(s)-M2 that results inthe activation of Cre recombinase expression. Cre recombinaseinactivates the RhCMV/HCMV vaccine vectors by catalyzing the excision ofthe region of the viral genome flanked by loxP sites (in this case,Rh52-Rh156). For simplicity, genes expressing rtTA2^(s)-M2 and tTS-kidas well as the gene expressing the heterologous pathogen antigen are notshown.

FIG. 17 shows another exemplary tetracycline-regulated RhCMV/HCMV‘safety’ vaccine vector. Such RhCMV/HCMV vaccine vectors are constructedby placing a gene essential for virus replication, in this example Rh70(HCMV homologue-UL44) DNA polymerase processivity factor, under controlof the Tet-inducible system described in FIG. 16. The Rh70HCMV homologue(UL44) was initially selected as this gene has been shown to beessential for CMV replication (Shenk, Proc. Natl. Acad. Sci. USA100:12396, 2003; Ripalti, J Virol. 69:2047, 1995). However, otheressential viral genes can be used as candidate genes for Tet-mediatedregulation. Regulation of Rh70 expression or expression of otheressential RhCMV/HCMV genes by the Tet-regulated system enables controlof virus replication by varying Dox level. To place the essential geneunder Dox control, the 5′ upstream region of the gene is replaced withthe tetO₇-CMV minimal promoter unit (white arrow). After inoculation ofanimals with Tet-regulated vectors in the presence of Dox, virusreplication can be inactivated simply by Dox withdrawal. Tet-regulatedvectors are constructed by E/T and Flp-based recombination as detailedin FIG. 15.

FIG. 18 shows yet another exemplary tetracycline-regulated RhCMV/HCMV‘safety’ vaccine vector. Such RhCMV/HCMV vaccine vectors are constructedcontaining a cytotoxic gene (CytoG) under control of the Tet-induciblesystem as detailed in FIG. 17. After inoculation of animals withTet-regulated vectors in the absence of Dox, virus replication can berapidly inactivated by Dox-mediated induction of the cytopathic generesulting in death of the vaccine vector-infected cell.

In further aspects, RhCMV and HCMV gene therapy vectors are provided.FIG. 19 schematically depicts construction of exemplary RhCMV and HCMVgene therapy vectors. Therapeutic gene(s) are inserted into RhCMV orHCMV BACs by E/T and Flp-mediated recombination. The schematic shows ageneralized strategy for insertion of an epitope-tagged therapeutic geneinto the non-coding region between rh213 and Rh214 of RhCMV. Thisstrategy can be similarly used for insertion of therapeutic genes atother defined sites within the RhCMV/HCMV genome. The gene encoding theepitope-tagged replacement gene is inserted into the BAC genome usingE/T recombination. Following selection of recombinant BACs on the basisof antibiotic resistance (in this case, Kan), the resistance gene isremoved by Flp-mediated recombination. Recombinant RhCMV and HCMV genetherapy vectors are reconstituted by transfection of recombinant BACsinto RhCMV/HCMV permissive cells (ThG, therapeutic gene; Tag, epitopetag; pA, polyadenylation site; Kan, kanamycin resistance gene forselection in bacteria).

Example 2 Construction of RhCMV/SIVmac239gag

The presence of the single functional LoxP site located in theintergenic region between Rh181 (US1) and Rh182 (US2) of RhCMVvLoxP wasexploited to utilize a Cre recombinase/LoxP system for construction ofrecombinant virus. For this approach, pSIVmac239gag plasmid was used asa source of template for PCR amplification of the SIVmac239gag cassette.The SIVmac239gag cassette contains the cellular EF1α promoter drivingexpression of the SIVmac239gag gene. The EF1α promoter is a highlyactive promoter that is constitutively active in all cell types testedand is expected to result in high cell type independent expression ofthe gag gene. PCR amplification was performed using primers designed toincorporate a single LoxP site at either end of the amplifiedSIVmac239gag cassette. For recombination, the PCR product containing theSIVmac239gag cassette flanked by LoxP sites was transfected into RMfibroblasts. At 24 hours post-transfection, these cells were infectedwith RhCMVvLoxP at a multiplicity of infection (MOI) of 1. The infectionwas allowed to progress until extensive cytopathic effect was observed.At this time, virus-infected cells were harvested and used to infectfresh fibroblasts, which were then overlayed with agarose to preventviral spread through the culture. After approximately two weeks,individual viral plaques were picked, and each plaque was used to infectfresh fibroblasts. Total cell lysates were then screened for thepresence of the SIVmac239gag gene by PCR. SIVmac239gag-positive celllysates were then sonicated, serial diluted and used to infect freshfibroblasts. The process was repeated three times, after whichplaque-purified virus clones were screened for gag gene expression bynorthern blot analysis of total RNA obtained from infected cells. Thepresence of gag protein expression was confirmed by western analysis aswell as immunofluorescence in endothelial cells (EC) andmonocyte-derived macrophages (MDM). The entire SIVmac239gag cassette wassequenced to confirm sequence integrity of the inserted cassette. Thegrowth kinetics of gag-positive clones was compared to WT virus and asingle RhCMV/SIVmac239gag recombinant virus with WT growthcharacteristics and high levels of gag expression was selected.

At day 224 post-primary infection, RhCMV/SIVmac239gag was subcutaneouslyadministered to a cohort of 4 RhCMV-seropositive RM. As previouslyobserved in re-infection studies, real-time PCR did not detect RhCMV,either WT or RhCMV/SIVmac239gag, in blood or lung lavage mononuclearcells at any time point following viral inoculation. However, urine fromday 127 post re-infection was weakly positive for gag expression byELISA suggesting the presence of RhCMV/SIVmac239gag. These samples wereco-cultured to isolate RhCMV, and these in vivo-derived viralpreparations were assessed for gag expression by western blot. As shownin FIG. 8, RhCMV co-cultures from all 4 animals expressed gag,definitively establishing the presence of RhCMV/SIVmac239gag virus atthese epithelial secretory sites. Retrospective analysis of urine atearlier post-re-infection time points revealed the presence of thegag-expressing RhCMV vector in urine by day 7 in one RM and by day 21 inthe other 3 animals. Moreover, gag-expressing RhCMV was also present insaliva, and remained present in urine at least through day 237 postre-infection. Significantly, re-infection of this RhCMV/SIVmac239gagclone induced a mucosally-oriented, gag-specific CD4+ and CD8+ T cellresponse, as well as a gag-specific antibody response (see FIGS. 10, 11and 12). These data unequivocally demonstrate that CMV is capable ofre-infecting immune subjects, effectively competing with pre-existent WTvirus, and establishing infection at normal sites within the host,despite strongly boosted cellular and humoral immunity directed at CMV.They also demonstrate the high in vivo stability of RhCMV vectorsexpressing exogenous heterologous antigens, suggesting that thesevectors may be able to persistently infect inoculated subjects andindefinitely maintain expression of the inserted, exogenousantigen-encoding genes. Finally, they demonstrate that recombinant RhCMVcan elicit both T cell and Ab responses to exogenous proteins in thesetting of re-infection, and thus has the potential to serve as aneffective vaccine vector in individuals with pre-existing CMV immunity.

Example 3 CMV Vectors Encoding Poliovirus VP1, TetanusC, Influenza H5N1Hemagglutinin and Ebola Virus Nucleoprotein (NP)

This example describes in vitro expression of heterologous antigensencoded by a recombinant murine CMV (MCMV) vector, and induction ofantigen-specific CD8+ T cells in vaccinated animals. CMVs show strictspecies specificity with most mammalian species having their own uniqueCMV. However, all CMVs share common characteristics of growth,cytopathology, tissue tropism and a capacity to establish persistent andlatent infection. The similarity in biology of CMVs has enabled the useof CMV infection of non-human animals, such as murine CMV infection ofmice and rhesus CMV infection of rhesus macaques, as in vivo models ofhuman disease. Thus, the MCMV mouse model is commonly used as an in vivomodel for HCMV. Accordingly, this example provides proof of concept foruse of recombinant CMV vectors (such as RhCMV and HCMV vectors) forexpression of heterologous antigens and use of the vectors for elicitingan immune response in a subject.

The MCMV BAC plasmid used in the following experiments to generaterecombinant MCMV vectors encoding a heterologous antigen has beendescribed (Wagner et al., J. Virol. 73:7056-7060, 1999).

DNA encoding poliovirus VP1 was PCR amplified from poliovirus nucleicacid and cloned into the MCMV BAC plasmid to generate pMCMV-VP1. The VP1protein was also tagged with a small epitope at the extremecarboxy-terminus to facilitate detection. The pMCMV-VP1 was transfectedinto murine embryo fibroblasts (MEFs) to reconstitute MCMV-VP1 virus.MEFs were infected with MCMV-VP1 virus. After approximately 3 days,cells were harvested and isolated protein was separated on apolyacrylamide gel. Western blotting was performed using an antibodyagainst the epitope tag. Based on epitope tag reactivity and predictedmolecular weight, the results demonstrated that poliovirus VP1 wasefficiently expressed in MEFs infected with MCMV-VP1. Aspects of thisembodiment of the invention are further described in Example 6.

The avian influenza (Viet04 strain) H5N1 HA was cloned into the MCMV BACplasmid to generate pMCMV-HA. The HA protein was epitope-tagged tofacilitate expression analysis. MCMV-HA virus was reconstituted asdescribed above. MCMV-HA virus was used to infect MEFs. Afterapproximately 3 days, cells were harvested and isolated protein wasseparated on a polyacrylamide gel. Western blotting was performed usingan antibody against the epitope tag. Based on epitope tag reactivity andpredicted molecular weight, the results demonstrated that HA wasefficiently expressed in MEFs infected with MCMV-HA.

A T cell epitope of Ebola virus NP was fused to non-essential MCMV geneIE2 to generate the construct pMCMV-EBOV-NP_(CTL). MCMV-EBOV-NP_(CTL)virus was reconstituted in MEFs. Ten mice were immunized i.p. withMCMV-EBOV-NP_(CTL) on day 1 and boosted at week 4. Splenocytes wereharvested at week 8 to evaluate the number of NP-specific CD8+ T cellsusing intracellular cytokine staining (ICS) Briefly, splenocytes wereincubated in the presence of target antigen (NP) and brefeldin A for 6hours. The percentage of NP-specific CD8+ T cells was determined by flowcytometry based on expression of effector cytokines. For individualmice, the percentage of total CD8+ T cells specific for NP ranged fromapproximately 1-10%, demonstrating that immunization withMCMV-EBOV-NP_(CTL) was extremely effective for eliciting an NP-specificT cell immune response. This embodiment of the invention is furtherdescribed in Example 7.

Summary: These results demonstrate that recombinant CMV vectors encodinga heterologous antigen (either the entire full-length protein or anindividual epitope) are capable of effectively expressing theheterologous antigen in infected cells, and eliciting a strong T cellimmune response to the heterologous antigen.

Example 4 Recombinant RhCMV Encoding Monkeypox Virus Antigen A35R

This example describes a recombinant RhCMV vector encoding the monkeypoxvirus antigen A35R and A35R-specific T cell responses in rhesus macaques(RM) vaccinated with the recombinant virus vector. RMs were vaccinatedwith RhCMV expressing A35R, and BAL fluid was collected at days 7, 14and 21 post-vaccination for analysis of T cells in the lung (anaccessible mucosal effector site). Both CD4+ and CD8+ central (CM) andeffector memory (EM) responses specific for A35R were assessed by ICS asdescribed above (but using A35R peptides). At day 14, a significantincrease in the number of CM and EM A35R-specific T-cells (both CD4+ andCD8+) was observed in the vaccinated animal.

Example 5

Generation of an effective SIV vaccine using an RhCMV vector design thatis highly attenuated and sufficiently safe for human translation isdescribed. One approach takes the most direct route to vectorattenuation by engineering generally replication-deficient vectors, butthe requirement for CMV vector replication for inducing a protectiveimmune response against SIV is still unclear. A complementary approachgenerates RhCMV/SIV vectors that are attenuated for replication only inspecific target tissues that are associated with CMV disease andshedding, but that can still induce SIV protective immune responses ifviral replication is a necessary component for this immunity. One of thekey characteristics of primary HCMV/RhCMV infection is that individualsshed virus from glandular tissue such as salivary gland as well asexcrete virus into the urine (Pass, R. F. 2001. Cytomegalovirus. InFields Virology. P. M. H. David M. Knipe, Diane E. Griffin, Robert A.Lamb Malcolm A. Martin, Bernard Roizman and Stephen E. Straus, editor.Philadelphia: Lippincott Williams & Wilkins. 2675-2705).

Infected epithelial cells lining the salivary gland ducts and urinarytract or kidney are considered to be persistent sources of virus inthese tissues. First, a RhCMV/SIV vector is engineered that has all ofthe known genes involved in epithelial cell tropism removed from theRhCMV backbone. Preventing virus replication in this cell type maysignificantly reduce shedding of RhCMV from these tissues as well aspathogenicity in RM without the loss of SIV protective immunity.Altering RhCMV epithelial cell tropism may significantly attenuate thevaccine vector in RM, but the possibility remains that in the context offetal infection the vector may still be able to disseminate and causeCNS disease. Therefore, an additional embodiment of the inventionrelates to ensuring safety of the vector by altering its ability toreplicate in CNS and myeloid tissue using a novel approach based ontissue-specific miRNAs to inactivate essential RhCMV genes necessary forproduction of infectious virus. The ultimate RhCMV/SIV vaccine generatedin these studies may include a combination of these tropism defectsdepending on the characteristics of the SIV-specific T cell responsesobserved with the individual tropism knockouts.

Generation and characterization of a RhCMV/SIV vector completelydeficient for replication in epithelial cells by deletion of tropismgenes. HCMV and RhCMV replicate in many different cell types includingepithelial and endothelial cells, macrophages, neurons, endothelialcells, as well as fibroblasts (which is the prototypic cell for growthof the virus in vitro). HCMV and RhCMV mutagenesis studies have revealeda number of viral genes that are nonessential for replication infibroblasts, but that determine tropism of the virus for other celltypes. For example, HCMV UL128-131A genes were observed to be criticalfor viral entry into epithelial and endothelial cells, but notfibroblasts as described earlier. The RhCMV vector used for constructionof RhCMV/SIV constructs (68-1), has mutations in the UL128-131A genecluster and displays decreased replication in RM retinal pigmentepithelial (RRPE) cells. This decreased replication in RRPE cellsresults from the loss of the RhCMV homologues of HCMV UL128 and thesecond exon of UL130. While 68-1 is still capable of infectingepithelial cells, repair of the UL128 and 130 mutations results in a 2-3log increase in viral replication in these cells. Consistent with thefunction of these genes in epithelial tropism, RhCMV 68-1 exhibitsdecreased shedding into urine and saliva from normal epithelial sites ofpersistent replication in healthy adult RMs. Importantly, this‘experiment of nature’ indicates that targeting of epithelial tropismgenes is an effective strategy by which to attenuate the ability of CMVvectors to shed while maintaining vaccine efficacy. The presence of 4additional epithelial cell tropism genes in RhCMV (Rh01 Rh159, Rh160,and, Rh203) may explain the ability of 68-1 to retain a residual abilityto replicate in epithelial cells. Mutation of these genes result insevere (>4 log decrease) to moderate (1-2 log decrease) replicationdefects in epithelial but not fibroblast cells. Since inactivation ofRhCMV UL128 and 130 has an effect on the ability of the virus to infectepithelial cells and shed from infected RMs, the elimination of theremaining epithelial cell tropism genes may completely abrogateepithelial cell infection and eliminate viral shedding. The eliminationof the epithelial cell tropism may severely attenuate pathogenicity ofthe virus in vivo, especially in the ability of the virus to causeepithelium-associated disease such as pneumonia. Therefore, embodimentsof the invention relate to a RhCMV/SIV vector that has decreasedreplication in epithelial cells by deletion of these 4 additionalepithelial tropism genes from the 68-1 RhCMV genome. This vector isdesignated RhCMVΔEpiCMAX/SIV(gag) or (retanef) and to be used for invivo studies.

The construction of a RhCMV/SIV vector deleted for all known epithelialtropism genes [designated RhCMVΔEpiCMAX/SIV(gag) or (retanef)] requiresmultiple rounds of linear recombination to individually target the fourremaining tropism genes within the 68-1 BAC genome (which is alreadydeleted for UL128 and UL130). At the same time, a cassette expressingSIV gag or retanef with a distinct epitope tag under control of the EF1αpromoter is reciprocally inserted into the genome during deletion of thefirst epithelial tropism open reading frame (ORF) (Rh01). To enablerepeated rounds of deletion, a modification to the standard two-steplinear recombination protocol that uses galactokinase (galK) as theselectable marker is used (Warming, S., Costantino, N., Court, D. L.,Jenkins, N. A., and Copeland, N. G. 2005. Simple and highly efficientBAC recombineering using galK selection. Nucleic Acids Res 33:e36).

Importantly, this modified system does not leave any cis-actingheterologous sequence DNA ‘scar’ within the BAC genome that wouldinterfere with subsequent rounds of recombination. In the firstrecombination step, recombinant bacteria (SW102) containing the RhCMVBAC (68-1) are transformed with a PCR product comprised of galK andkanamycin resistance (KanR) flanked by sequence homologous to thedesired site of recombination within the RhCMV genome. SW102 aregalactose auxotrophs (due to deletion of the galK gene from thegalactose operon), and growth on plates containing galactose as the onlysource of carbon and kanamycin enables selection for BAC clones thathave incorporated the PCR product (and galK/KanR) within the genome.

In the second recombination step, the galK/KanR cassette is replacedwith an oligonucleotide or PCR product with sequence homology to therecombination site flanking the desired deletion. Bacteria is grown onplates containing 2-deoxy-galactose, a substrate toxic in the presenceof galK, in the absence of kanamycin resulting in counter-selection forBAC clones that have lost the galK/KanR marker, and contain only thedesired deletion. This method is used to sequentially delete Rh159,Rh160, and, Rh203 until all of the epithelial cell tropism genes aredeleted from the RhCMV/SIV vector. In parallel to reduce the numbers ofanimals necessary to test the immunogenicity of the RhCMV/SIV vectorssimilar RhCMV vectors lacking the epithelial cell tropism genes butencoding SIV retanef with a V5 epitope tag expression cassette (2)instead of the gag expression cassette [RhCMVΔEpiCMAX/SIV(retanef) areconstructed.

Reconstitution of RhCMVΔEpiCMAX/SIV(gag) and (retanef) viruses: Toreconstitute viruses, BACs are transfected into primary rhesusfibroblasts (RFs). After approximately two weeks, plaques are expectedto become visible and virus is recovered. Virus is further purified bythree rounds of plaque purification to ensure removal of the BACcassette and elimination of any residual contamination with wildtypeRhCMV. Removal of the BAC-cassette is confirmed by PCR and Southern blotfrom genomic DNA isolated from recovered virus. Expression of gag andretanef protein expression is confirmed to ensure that target antigenexpression levels are comparable to WT vectors. Growth ofRhCMVΔEpiCMAX/SIV(gag) and (retanef) is compared to wildtype RhCMV forgrowth in both RFs (assessment of basic growth capacity) and epithelialcells.

In addition to RRPE, primary RM kidney epithelial cells may be used forthis characterization. Growth on macrophages and microvascularendothelial cells is also assessed since these genes may effect growthof the virus in these cell types. An RhCMV vector that retains normalgrowth in fibroblasts in vitro, but is maximally compromised for growthin epithelial cells will be tested for immunogenicity and attenuation inanimals. If viral replication is also altered in macrophages orendothelial cells, an embodiment of the invention relates to mutationsthat exhibit lack replication in epithelial cells alone. Since the fourindividual epithelial genes may be sequentially deleted in constructionof RhCMVΔEpiCMAX/SIV(gag) and (retanef), any vector that shows decreasedreplication in fibroblasts or additional non-epithelial cell types israpidly identified at the stage of its construction during thesubsequent in vitro growth characterization. The RhCMV vector that ismaximally deleted for the greatest number of the epithelial tropismORFs, without affecting fibroblast growth is designatedRhCMVΔEpiCMAX/SIV(gag) or (retanef) and used for the in vivo RM studies.

Embodiments of the invention relate to the generation andcharacterization of RhCMV/SIV vectors that are deficient for replicationin epithelial, neuronal and myeloid tissue by insertion oftissue-specific miRNA target sequences into the 3′UTRs of essentialRhCMV genes.

One of the main concerns of using a wildtype HCMV-based HIV vaccine isthe potential for disease in immunocompromised fetuses, children andadults. HCMV can cause a variety of diseases in these individualsincluding CNS abnormalities, peripheral neuropathy, retinitis, andpneumonia (Pass, R. F. 2001. Cytomegalovirus. In Fields Virology. P. M.H. David M. Knipe, Diane E. Griffin, Robert A. Lamb Malcolm A. Martin,Bernard Roizman and Stephen E. Straus, editor. Philadelphia: LippincottWilliams & Wilkins. 2675-2705). The loss of epithelial cell tropism inRhCMV vectors may attenuate the development of pneumonia and retinitisthough CNS disease remains a major concern. CNS disease is a majorconcern during congenital infection that can lead to hydrocephalous,malformation of the brain and deafness. Insertion of tissue specificmiRNA target sequences into the poliovirus genome was shown to beextraordinarily effective in attenuating viral replication in the brainand mortality in mice providing a novel approach for vaccinedevelopment. Similarly, insertion of miRNA targets into the influenzagenome also inactivates virus in tissues.

Some embodiments of the invention relate to specifically preventing CMVreplication in macrophages by targeting an essential gene of the viruswith an hematopoietic miRNA. The functionality of this approach wasdemonstrated for MCMV where expression of the essential IE3 gene wasknocked down by miR 143 3p (FIGS. 21-25). The utility of this approachfor targeting mRNA of essential genes of CMV for destruction by cellularmiRNAs is described herein. Importantly, miRNAs are highly conservedbetween mice and humans (and therefore RM) making this approach feasiblefor other species (Larke, R. P., Wheatley, E., Saigal, S., andChernesky, M. A. 1980. Congenital cytomegalovirus infection in an urbanCanadian community. J Infect Dis 142:647-653). A similar approach toconstruct CMV vectors attenuated for growth in the CNS by the use ofneuron-specific miRNAs is utilized.

Another characteristic of CMV disease is dissemination of the virus intoorgans and this is considered to be mediated by macrophages. Macrophagesand endothelial cells are considered to be sites of CMV persistence inthe host so elimination of one of the reservoirs of the virus shouldstill enable virus persistence (Jarvis, M. A., and Nelson, J. A. 2002.Human cytomegalovirus persistence and latency in endothelial cells andmacrophages. Curr Opin Microbiol 5:403-407). Therefore, some embodimentsof the invention relate to targeting myeloid replication to prevent CMVsystemic dissemination. An optimal CMV vector candidate will havewildtype immunogenicity, while being maximally attenuated forreplication in the widest diversity of different cell types. CMV tissueknockout vectors targeted to prevent replication in the followingtissues are generated: CNS, hematopoietic, CNS/epithelial,CNS/hematopoietic, epithelial/hematopoietic, andCNS/hematopoietic/epithelial.

In vitro tissue tropism characteristics of the CNS (RhCMVΔC/SIV) andhematopoietic (RhCMVΔH/SIV) tissue knockout viruses are generated andtested. The number of RM for immunogenicity studies may be conserved bymaking tissue tropism knockouts with both SIV gag, RhCMV/SIV(gag), andthe SIV rev, tat and nef fusion protein, retanef, RhCMV/SIV(retanef) aspreviously described. The construction of these vectors is as follows:

RhCMVΔC/SIV(gag) and (retanef): To generate a RhCMV/SIV vaccine thatcannot replicate in the CNS, 4 repeated target sequences (four 21mers)with exact complementarity to the neuronal miR-124 into the 3′UTRsequence of the RhCMV essential gene IE2 gene (Rh156 Ex5) of theRhCMV/SIV vectors are inserted. miR-124 in mouse, RM, and human areconserved at the nucleotide level (5′UAAGGCACGCGGUGAAUGCC3′ (SEQ ID NO:13)) (as further described on the website of miRBase, the microRNAdatabase). In order to ensure a complete knockout of virus replicationin this tissue, the miR-124 target sequences are inserted into the 3′UTR of DNA helicase gene (Rh142). For IE2 the miR-124 target sequencesare inserted 18 base pairs down stream of the ORF, effectively themid-point of the 3′UTR based on the predicted AATAAA polyadenylationsignal or 78 base pairs down stream of Rh142 using BAC technologydescribed above.

In each case overlapping ORFs are avoided to minimize the possibility ofnon-specific attenuation of the virus. Once constructed, each of theviruses are tested for gene expression levels of the target gene andattenuation of viral growth in fibroblast cells stably transfected withinducible constructs expressing each of the tissue specific miRNAs. RNAexpression levels are tested by RT-PCR while protein expression levelsof the target genes are monitored by western blot analysis. Virus growthis determined by both one-step and multi-step growth curves, comparingthe viruses containing the miRNA target versus the control viruses inprimary cultures of RM fetal neuronal cultures in comparison to RFs.HCMV has been shown to replicate in neuronal cells in vitro (Luo, M. H.,Schwartz, P. H., and Fortunato, E. A. 2008. Neonatal neural progenitorcells and their neuronal and glial cell derivatives are fully permissivefor human cytomegalovirus infection. J Virol 82:9994-10007). Primaryneuronal cultures from fetal RM brain tissue to isolate brain capillaryendothelial cells are available with the Applicants. Viruses thatexhibit growth in RFs but not neuronal cultures are tested forpathogenesis in as well as the T cell response.

RhCMVΔH/SIV(gag) and (retanef): To generate a RhCMV/SIV vector thatcannot replicate in macrophages, four 21mers with exact complementarityto the hematopoietic cell miR-143-3p into the 3′ UTRs of two RhCMVessential late genes are inserted: gH(Rh104) the essential glycoproteinreceptor, and minor capsid protein (Rh117). The sequence of miR-143-3Pfor mouse, RM, and human are conserved at the nucleotide level(5′UGUAGUGUUUCCUACUUUAUGGA3′ (SEQ ID NO: 14)) (as further described onthe website of miRBase, the microRNA database). For gH the miR-143-3ptarget site is positioned 114 bp downstream of the ORF before the poly Asequence, and for Rh117 the target sequence is 30 bp downstream of theORF. Since Rh111, 112, 114 and 118 use the same AATAAA polyadenylationsignal as Rh117, all of these transcripts are expected to be knocked outin macrophages thereby increasing the effectiveness of the viralreplication block.

The RhCMV essential late genes are targeted to prevent production ofinfectious virus, but permit expression of the SIV gag and retanefantigens in macrophages to allow appropriate gag and retanef antigenpresentation to preserve vaccine vector immunogenicity. Viruses thatexhibit growth in RF but not macrophages are tested for pathogenesis aswell as T cell response in. Macrophages are prepared from RM peripheralblood as previously described (Söderberg-Naucler, C., Fish, K. N., andNelson, J. A. 1997. Reactivation of latent human cytomegalovirus byallogeneic stimulation of blood cells from healthy donors. Cell91:119-126).

Along with testing single tropism deficient RhCMV/SIV vectors,embodiments of the invention relate to constructing the multiple tropismdeficient RhCMV vectors with gag and retanef combining the features ofthe epithelial, CNS and macrophage deficient tropisms. Such vectors andregions of growth deficiency are listed in Table 3:

TABLE 3 Vector construction and in vitro characterization Vector nameGrowth Deficiency (Single Tropism Deficient) RhCMVΔEpiCMAX/SIV(gag) and(retanef) Epithelium RhCMVΔC/SIV(gag) and (retanef) CNS RhCMVΔH/SIV(gag)and (retanef) Hematopoietic (Multiple Tropism Deficient)RhCMVΔEpiCMAXΔC/SIV(gag) and (retanef): Epithelium/CNS RhCMVΔCΔH/SIV(gag) and (retanef) CNS/Hematopoietic RhCMVΔEpiCMAXΔH/SIV (gag) and(retanef): Epithelium/ Hematopoietic RhCMVΔEpiCMAXΔCΔH/SIV(gag) and(retanef): Epithelium/ Hematopoietic/ CNS

Each of the multiple tropism deficient RhCMV/SIV vectors are tested invitro for growth in RF in comparison to neuronal, epithelial,endothelial and macrophage cultures as described above.

Further embodiments of the invention relate to comparing the in vivoreplication dynamics, persistence, secretion, immunogenicity, andpathogenicity of tropism-modified RhCMV/SIV vectors with that ofwildtype RhCMV/SIV vectors.

During congenital infection, the CNS is a major target tissue resultingin hearing loss in the infant. In the immunosuppressed adult, directinfection of epithelial and endothelial cells results in GI, lung andretinal disease, respectively. In all cases myeloid cells are believedto be critical for dissemination of the virus through the host (Sopper,S., Nierwetberg, D., Halbach, A., Sauer, U., Scheller, C., Stahl-Hennig,C., Matz-Rensing, K., Schafer, F., Schneider, T., ter Meulen, V., et al.2003. Impact of simian immunodeficiency virus (SIV) infection onlymphocyte numbers and T-cell turnover in different organs of rhesusmonkeys. Blood 101:1213-1219). Using vectors that are deficient in theirability to replicate in multiple different cell types/tissues may impactthe potential for CMV to cause multiple forms of virus-associateddisease in all immunocompromised target populations (fetus and adult).Epithelial cells of the oral mucosa, breast and kidney are also majorsites of virus shedding. The epithelial tropism defective vectors areadditionally expected to impact shedding of virus into saliva and urineand breast milk. Decreased shedding is important for preventingenvironmental spread of vectors from initial vaccinees to non-vaccinatedcontacts. Embodiments of the invention relate to the identification ofan optimal modified vector that is selected for efficacy trials. Theselected vector is chosen based on its ability to induce and maintain aT_(EM)-biased immune response against their encoded SIV transgenecomparable to wildtype RhCMV vectors, whilst showing decreasedreplication in tissues targeted by CMV during disease, and shedding (asdescribed below).

A two-stage strategy for construction and analysis of tropism-modifiedvectors (as listed in Table 3). Single-tropism modified vectors:Embodiments of the invention relate to vectors are constructed that areindividually deficient for replication in either epithelial cells[RhCMVΔEpiCMAX/SIV(gag) and (retanef)], CNS [RhCMVΔC/SIV(gag) and(retanef)] or myeloid (hematopoietic) cells RhCMVΔH/SIV(gag) and(retanef)]. Following in vitro characterization (see SA1), vectors thatdisplay the desired modified tropism phenotype are assessed forimmunogenicity in adult/juvenile RMs and pathogenicity/tropism in thefetal model. Any single-tropism modified vector that shows unanticipatedin vitro growth defects is rejected and is not further characterized.Similarly, lack of immunogenicity in adult RMs (i.e., the inability toinduce a SIV-specific T cell response), or undesired in vivo tropism(based on analysis in the fetal model) will result in vector rejection.

Multiple-tropism modified vectors: Further embodiments of the inventionrelate to the construction of multiple-tropism modified vectors based onsingle-tropism vectors that show desired in vitro or in vivo modifiedtropism. After determining single-tropism modified vectors immunogenicin adult RMs and showing the desired in vivo tissue tropism in the fetalmodel, multiple-tropism vectors built on these genetic backgrounds, thatshow the desired multi-tissue in vitro tropism, will be analyzed in vivoin a comparable fashion to the single-tropism vectors. This group ofmultiple-tropism vectors comprises four constructs that are deficientfor replication in the following multiple tissues: epithelial andmyeloid cells [RhCMVΔEpiCMAXΔC/SIV(gag) and (retanef)], epithelial cellsand CNS [RhCMVΔEpiCMAXΔC/SIV(gag) and (retanef)], myeloid cells and CNS[RhCMVΔHΔC/SIV(gag) and (retanef)], and all three target tissues[RhCMVΔEpiCMAXΔHΔC/SIV(gag) and (retanef)] (Table 3).

Aspects of the invention relate to the assessment of RhCMV/SIV vectorimmunogenicity. Initial immunological analysis is performed in groups of3 RMs/vector (FIG. 26). Each group receives both gag and retanefversions of each vector as a mixture in a single inoculum. Vaccinationof sero+ RMs with wildtype RhCMV/SIV vectors results in induction of adetectable T cell response against the SIV transgene in 100% of animalsby 3 weeks post-infection as described previously. Any vector pair(individually encoding gag and retanef) that fails to induce adetectable T cell response against SIVgag or retanef by the time ofboost at week 15 will considered to have “failed” on the basis ofimmunogenicity and need not be further characterized. For immunogenicvectors, all 3 RMs are boosted at week 15 with the gag-expressing vectoronly, thereby enabling characterization of both ‘boosted’ (gag) andnon-boosted (retanef) responses. At the same time, 3 more RMs arerecruited into the cohort and treated in an identical fashion as thefirst 3 animals, so as to supply sufficient statistical power forcomplete immunological characterization. All RM are followedimmunologically and virologically for a total of 45 weeks post-initialvaccination (see FIG. 2 and below).

All RM are necropsied at week 48 for systemic quantification ofSIV-specific T cell responses. A total of 24 RMs are available forimmunogenicity testing of tropism-modified vectors (in addition to acontrol cohort of 6 RMs given wt RhCMV/gag×2 and RhCMV/retanef×1). Thisnumber of RM translates into several potential different in vivo testingscenarios depending on the final number of tropism-modified vectors that‘pass muster’ and need complete evaluation. For example, 5 vectors mayinitially be tested in 3 RMs each, of which 3 are selected for completeimmunological testing in an additional 3 RMs (to give a full complementof 6 RMs for each of the 3 vectors). Alternatively, 6 vectors mayinitially be tested in 3 RMs each, of which 2 of 6 (the most promisingin terms of immunogenicity and/or attenuation) are then selected forcomplete immunological testing in an additional 3 RMs (to make a fullcomplement of 6 RMs for each of the 2 vectors).

CD4⁺ and CD8⁺ T cell responses to the SIV proteins (gag andrev/nef/tat), negative control proteins (TB Ag85B protein), and RhCMV IE(+control) are quantified by cytokine flow cytometry (CFC) usingoverlapping, consecutive 15-mer peptide mixes comprising these proteinsas previously described in Hansen, S. G., Vieville, C., Whizin, N.,Coyne-Johnson, L., Siess, D. C., Drummond, D. D., Legasse, A. W.,Axthelm, M. K., Oswald, K., Trubey, C. M., et al. 2009. Effector memoryT cell responses are associated with protection of rhesus monkeys frommucosal simian immunodeficiency virus challenge. Nat Med 15:293-299.

This assay is applied to PBMC, lymph node (LN) and bronchioalveolarlavage lymphocytes [BAL, an easily accessible effector site, where RhCMVvector elicited responses are highly enriched] per the animal protocol(FIG. 26), and at necropsy to cell preparations obtained from at least 6different peripheral LN groups, 3 different mesenteric LN groups, BAL,bone marrow, spleen, liver, jejunal/ileal/colonic mucosa, genitalmucosa, and tonsil/adenoids. Routine assessment uses stainingcombination #1 (Table 4) in which the responding cells are delineated byup-regulation of CD69 and intracellular expression of TNF, γ-IFN andIL-2 (alone or in any combination), and simultaneously classified totheir differentiation state by expression of CD28 vs. CCR7 (Picker, L.J., Reed-Inderbitzin, E. F., Hagen, S. I., Edgar, J. B., Hansen, S. G.,Legasse, A., Planer, S., Piatak, M., Jr., Lifson, J. D., Maino, V. C.,et al. 2006. IL-15 induces CD4 effector memory T cell production andtissue emigration in nonhuman primates. J Clin Invest 116:1514-1524.).Staining combination #2 is designed to detect changes in activation andproliferative status of Ag-specific T cells (defined by CD69, TNF andγ-IFN induction), and is used to precisely follow the response ofestablished SIV-specific T cell populations to in vivo Ag exposure afterboosting. Staining combination #3 analyzes MIP-1β and cytotoxicdegranulation (CD107 externalization) in the context of memory subsets,and is selectively used to extend functional analysis of the response inPBMC at least once for each response prior to necropsy.

Embodiments of the invention relate to CFC assays being performed and inaddition, relate to the gag- and rev/nef/tat-specific T cell responsesthat may be transcriptionally analyzed in PBMC and at necropsy inselected tissues by Ag-stimulated microarray analysis. In this approach,transcriptional changes associated with specific Ag versus controlstimulation are compared at 3 and 12 hours post-stimulation to provide adetailed assessment of the functional programs of Ag-responding T cells.

TABLE 4 Standard CFC analyses for monitoring RhCMV/SIV vectorimmunogenicity Ag-Specific Response Assays: Routine Staining Panel PBACy FITC PE ECD TrR PC7 APC A700 AC7 Comments 1 CCR7 CD4 TNF IL2 CD28*CD69 CD95 IFNγ CD3 CD8 TNF, IL2, IFNγ; central/effector memory 2 HLA-DRCD4 Ki-67 PD-1 CD25 CD69 TNF IFNγ CD3 CD8 TNF, IFNγ; in vivo activation3 CCR7 CD4 CD107** MIP-1β CD28* CD69 CD95 TNF CD3 CD8 TNF, MIP-1β;degranulation; cent./effec. mem. Notes: PB = Pacific Blue: ACy = AmCyan;F = fluorescein; PE = phycoarythrin; ECD = PE-Texas Red; TR = True Red(PerCP-Cy5.5); APC = allophycocyanin; A700 = Alexa700; PC7 = PE-Cy7; AC7= APC-Cy7; intracellular Ags are in bold; *CD28 conjugate provided asco-stimulation along with CD49 mAb in stimulation culture; CD107conjugates also included with cells during incubation.

The success of each modified vector with respect to immunogenicity isprimarily judged by the magnitude of total SIV-specific CD4⁺ and CD8⁺ Tcells in PBMC, LN and BAL during the vaccine phase (peak and plateau)and the overall size of the SIV-specific CD4⁺ and CD8⁺ T cellpopulations in lymphoid tissues and effector sites at necropsy. Overallsystemic response magnitude at necropsy is evaluated by determining theaverage frequency of SIV-specific T cells in each tissue at necropsy,and by using previous systemic T cell counting data (Halbach, A.,Nierwetberg, D., Muller, J. G., Sauer, U., Kerkau, T., Stolte, N.,Hofmann, P., Czub, S., ter Meulen, V., and Sopper, S. 2000. Totalnumbers of lymphocyte subsets in different lymph node regions ofuninfected and SIV-infected macaques. J Med Primatol 29:148-157; Sopper,S., Nierwetberg, D., Halbach, A., Sauer, U., Scheller, C., Stahl-Hennig,C., Matz-Rensing, K., Schafer, F., Schneider, T., ter Meulen, V., et al.2003. Impact of simian immunodeficiency virus (SW) infection onlymphocyte numbers and T-cell turnover in different organs of rhesusmonkeys. Blood 101:1213-1219.) to calculate the total numbers ofSIV-specific T cells in all sampled locations. These data arestatistically analyzed using Wilcoxon rank sum test. Since RhCMV/SIVvector-elicited CD8⁺ T cell responses correlate most closely to efficacyand preference will be given to vectors eliciting the largest responsesin this lineage, particularly when these populations are localized inSIV target cell-rich effector sites. Secondary criteria is thefunctional, phenotypic and transcriptional characteristics of thesteady-state SW-specific T cell responses in chronic phase, withmodified RhCMV/SIV vector-elicited T cell responses that most closelyrecapitulate wildtype RhCMV/SIV vector-elicited responses with respectto T_(EM) phenotypic, functional and transcriptional attributes havingpriority.

Assessment of RhCMV/SIV vector shedding: Virologic quantification ofvectors shed into the saliva and urine determines whether vectorsexhibit decreased shedding from these sites. Titers of RhCMV vectors insaliva and urine, as well as blood is determined by quantitative RT-PCRby using either gag or retanef cassette specific primers. RhCMV-specificprimers directed against RhCMV IE are used to quantify total RhCMVlevels (irrespective of SIV transgene). Co-culture of either saliva orurine with permissive RFs followed by western analysis against eithergag or retanef is used as additional maximally sensitive, butnon-quantitative measure of vector detection.

Other embodiments of the invention relate to the assessment RhCMV/SIVfetal pathogenicity/tissue tropism: Vectors are also assessed forpathogenicity/tissue tropism in the fetal model. In vivo cellulartropism, as well as level of pathogenicity are critical parametersmeasured during analysis in this model. Genes are known to bedifferentially regulated in the fetus compared to the adult (Merkerova,M., Vasikova, A., Belickova, M., and Bruchova, H. MicroRNA expressionprofiles in umbilical cord blood cell lineages. Stem Cells Dev19:17-26). Fetuses are inoculated with each RhCMV/SIV vector asdescribed previously. The formalin-fixed/paraffin-embedded/H&E-stainedtissue sections for RhCMV sequelae are evaluated, using standardmethodologies. Tissues sections are examined in a blinded fashion andscored on a scale of normal to mild to severe. The pathogenic potentialof vectors is assessed by using ultrasound and morphometrics (Chang, W.L., Tarantal, A. F., Zhou, S. S., Borowsky, A. D., and Barry, P. A.2002. A recombinant rhesus cytomegalovirus expressing enhanced greenfluorescent protein retains the wild-type phenotype and pathogenicity infetal macaques. J Virol 76:9493-9504).

Analysis of vector distribution in different tissues by quantitativeRT-PCR, and immunohistochemistry using antibodies directed againstcellular markers, enables determination of whether tropism phenotypespersist in vivo. Antibodies used in these studies are directed againstSIV and RhCMV antigens and RM cellular markers beta-III tubulin orneuronal marker neuronal nuclei (immature and mature neurons), glialfibrillary acid protein (astrocytes), vimentin (fibroblast), vWF or CD31(endothelial cells), cytokeratin or zonula occludens-1 (epithelialcells), smooth muscle actin (smooth muscle cells), CD68 (macrophage),CD3 (T cells), and CD20 (B cells). Immunohistochemical staining forRhCMV IE1 is performed according to published protocols (Abel, K.,Strelow, L., Yue, Y., Eberhardt, M. K., Schmidt, K. A., and Barry, P. A.2008. A heterologous DNA prime/protein boost immunization strategy forrhesus cytomegalovirus. Vaccine 26:6013-6025; Lockridge, K. M., Sequar,G., Zhou, S. S., Yue, Y., Mandell, C. P., and Barry, P. A. 1999.Pathogenesis of experimental rhesus cytomegalovirus infection. J Virol73:9576-9583; Bissel, S. J., Wang, G., Ghosh, M., Reinhart, T. A.,Capuano, S., 3rd, Stefano Cole, K., Murphey-Corb, M., Piatak Jr, M.,Jr., Lifson, J. D., and Wiley, C. A. 2002. Macrophages RelatePresynaptic and Postsynaptic Damage in Simian Immunodeficiency VirusEncephalitis. Am J Pathol 160:927-941; Sequar, G., Britt, W. J.,Lakeman, F. D., Lockridge, K. M., Tarara, R. P., Canfield, D. R., Zhou,S. S., Gardner, M. B., and Barry, P. A. 2002. Experimental coinfectionof rhesus macaques with rhesus cytomegalovirus and simianimmunodeficiency virus: pathogenesis. J Virol 76:7661-7671).

Tissue sections are scored for IE1 staining on a flexible scale of noneto minimal (isolated individual cells) to severe (extensive focal areasof staining) Staining protocols are done according to published andunpublished protocols as described in Batchelder, C. A., Lee, C. C.,Matsell, D. G., Yoder, M. C., and Tarantal, A. F. 2009. Renal ontogenyin the rhesus monkey (Macaca mulatta) and directed differentiation ofhuman embryonic stem cells towards kidney precursors. Differentiation78:45-56; Abenes, G., Lee, M., Haghjoo, E., Tong, T., Zhan, X., and Liu,F. 2001. Murine cytomegalovirus open reading frame M27 plays animportant role in growth and virulence in mice. J Virol 75:1697-1707;Carlson, J. R., Chang, W. L., Zhou, S. S., Tarantal, A. F., and Barry,P. A. 2005. Rhesus brain microvascular endothelial cells are permissivefor rhesus cytomegalovirus infection. J Gen Virol 86:545-549; Mazumdar,K., Alvarez, X., Borda, J. T., Dufour, J., Martin, E., Bethune, M. T.,Khosla, C., and Sestak, K. Visualization of transepithelial passage ofthe immunogenic 33-residue peptide from alpha-2 gliadin ingluten-sensitive macaques. PLoS One 5:e10228; Orzechowska, B. U.,Manoharan, M., Sprague, J., Estep, R. D., Axthelm, M. K., and Wong, S.W. 2009. Viral interleukin-6 encoded by rhesus macaque rhadinovirus isassociated with lymphoproliferative disorder (LPD). J Med Primatol 38Suppl 1:2-7). All antibodies are directed to human antigens but haveproven cross-reactivity with the RM orthologue.

Embodiments of the invention relate to the assessment of Efficacyagainst SIV Challenge. Based on all immunological, virological and fetalpathogenic/tropism characteristics, the most promising vectors areselected for use in efficacy trials in a low-dose intra-rectal SIVmac239challenge model. For these efficacy trials, the remaining SIV constructsfor a selected vector design (i.e., expressing env, pol I and II) areconstructed so as to have a complete vector set for efficacy assessment.Criteria for vector selection for assessment in efficacy trials and theadult immunosuppression model are primarily based on levels ofSIV-specific CD8+ T_(EM) cells at effector mucosal tissue sites, as wellas systemic immunogenicity, fetal pathogenicity/tissue tropism profileand shedding. Immunologically, an optimal vector induces and maintainscomparable or higher levels of T_(EM) biased responses in effectortissues against the SIV inserts. Virologically, the optimal vector has aminimal level of pathogenicity in the fetal pathogenicity model, andshows reduced shedding into saliva and urine.

Embodiments of the invention relate to pathogenicity testing in theAdult Immunosuppression Model: Gag-expressing versions of vectorsselected for efficacy trials are analyzed for pathogenicity in the adultimmune suppression model. This model uses iatrogenic immune suppressionof adult sero-negative animals undergoing primary RhCMV infection viathe IV route. Importantly, this model enables characterization of thefull pathogenic potential of RhCMV vectors in the primary anticipatedtarget population (i.e., young human adults). All RM go to necropsy forassessment of vector pathology and tissue tropism, as described abovefor fetal model. These data extend evaluation of vector safety andprovide additional criteria for selection of CMV vector designs forhuman translation.

Embodiments of the invention relate to the generation andcharacterization HCMV/HIV vectors corresponding to tropism-modifiedRhCMV/SIV vectors. HCMV/HIV vectors are constructed based on theRhCMV/SIV tropism deficient vectors that demonstrate the greatestattenuation without loss of SIV immunogenicity in RM described above.HCMV versions are constructed containing the same genetic modificationsas the selected RhCMV vectors exhibit a comparable phenotype in vitro.This aspect of the invention relates to the construction of the HCMV/HIVvector rather than the construction of an HCMV/HIV vaccine that would besuitable for use in clinical studies. Although most of the RhCMV genesare functionally equivalent, HCMV does have some epithelial tropismgenes that appear to be different from RhCMV (Dunn, W., Chou, C., Li,H., Hai, R., Patterson, D., Stolc, V., Zhu, H., and Liu, F. 2003.Functional profiling of a human cytomegalovirus genome. Proc Natl AcadSci USA 100:14223-14228.).

Although the RhCMV epithelial cell tropic genes rhUL128-131, Rh01,Rh159, Rh160, and, Rh203 have homologues in HCMV (UL128-131A, TRL,UL148, UL132, and US 22, respectively), only UL128-131A has beenidentified as an epithelial cell tropism gene in HCMV. The UL128-131Alocus from HCMV is not deleted since deletion of these genes alsoeliminates infection of endothelial cells and macrophages. However,targeted mutagenesis of HCMV revealed that mutation of UL64 and US29significantly reduced growth of HCMV in epithelial but not endothelialor human fibroblast (HF) cells as described previously. Therefore in thegeneration of the epithelial tropism-deficient virus these genes areideal to target for deletion in the generation of an epithelial celltropism-deficient HCMV vaccine. Translating the CNS and macrophagetropism-deficient RhCMV into HCMV is more straightforward since thegenes selected for miRNA targeting are essential genes in both viruses.HCMV tropism deficient vectors are tested for their ability to replicatein epithelial, neuronal, endothelial and macrophage cultures in vitro toensure the tropism defect. Importantly, these studies provide theconstruction ‘blue-print’ for the final HCMV/HIV vector to be used forclinical studies.

HCMV/HIV tropism deficient vaccine vectors are designed based on optimalattenuation and immunogenicity properties. 3-4 HCMV/HIV vaccine vectorsthat have combinations of altered tropism properties may be generated.The HCMV/HIV vaccines are constructed using the HCMV strain TR as thegenetic backbone (Murphy, E., Yu, D., Grimwood, J., Schmutz, J.,Dickson, M., Jarvis, M. A., Hahn, G., Nelson, J. A., Myers, R. M., andShenk, T. E. 2003. Coding potential of laboratory and clinical strainsof human cytomegalovirus. Proc Natl Acad Sci USA 100:14976-14981). ThisHCMV strain is a clinical isolate cloned as an infectious BAC thatmaintains diverse tropism for multiple cell types.

For epithelial tropism-deficient viruses HCMV genes known to be requiredfor growth in these cell types are first targeted, including the UL64and US29 (see above). Single and double deletion mutants of UL64 andUS29 are generated using BAC technology. Whether the single or doubleUL64/US29 HCMV mutants exhibit restricted growth in epithelial cells(human retinal epithelial cells and Caco 2 cells) but not endothelial orHF cells is determined. The miRNA knockout strategy for inactivation ofessential genes in CNS and macrophages is constructed using the approachdescribed above with RhCMV. A combination of the tropism deficientphenotypes is incorporated into one or two vectors with the RhCMV/SIVmultiple-tropism modified vectors. To determine whether the HCMV/HIVvectors exhibit the tropism defects predicted from the RhCMV/SIVstudies, the growth properties of these viruses in a variety ofdifferent cell types including HF, epithelial and endothelial cells,primary neuronal cultures and macrophages are determined. The cell typesincluding multiple micro- and macro-vascular endothelial cells,epithelial cells (retinal epithelial cells, Caco 2 cells), neuronalcells (SY5Y and SKMN cells), and primary macrophages generated fromperipheral blood mononuclear cells (57) that are able to sustain HCMVreplication are available to the Applicants. Single- and multi-stepgrowth curves are performed with these cells to compare the replicationefficiency of the HCMV/HIV tropism deficient vectors in comparison towildtype virus.

Example 6

Development of a novel, non-reverting, single-dose oral polio vaccine toreplace OPV. This example describes the use of the highly immunogeniccytomegalovirus (CMV)-based vaccine platform to develop a polio vaccinethat will induce protective immunity against all the 3 poliovirusserotypes, but has no ability to ‘revert’ into a pathogenic form ofpoliovirus.

Background: Vaccination with either the ‘killed’, inactivated poliovaccine (IPV) or ‘live’ attenuated oral polio vaccine (OPV) has resultedin a dramatic decrease in the incidence of polio-associated flaccidparalytic paralysis worldwide. Due to a number of characteristics (suchas low cost of production, oral administration and induction of mucosalimmunity) OPV was selected over IPV for the Global Polio EradicationInitiative (GPEI) in 1988. Since initiation of the GPEI program, OPVvaccination has resulted in a dramatic reduction in polio-associatedparalysis from >350,000 cases in 125 countries to <100 in 4 endemiccountries (as of March, 2011). With global eradication of wildtypepoliovirus imminent, followed by the planned stoppage of vaccination,‘reversion’ of OPV into paralytic forms of poliovirus becomes the lastremaining hurdle. OPV reversion results from genetic mutation ofresidual circulating ‘live’ OPV into a paralytic form of the virus, andis the sole reason for the need of an ‘endgame’ strategy.

The capacity of OPV reversion to cause disease out-breaks is a‘real-world’ problem, which is already occurring in developing countrieswith poor vaccine coverage. Implementation of ‘killed’ IPV vaccinationhas been proposed as one possible endgame strategy to tackle OPVreversion, with IPV vaccination being maintained until OPV is no longercirculating within the human population. Although this strategy may bepotentially effective, the cost of IPV vaccination (which has an highcost of production, and requires a needle and trained medical staff foradministration) makes this approach prohibitively expensive for use inpoorer nations. Embodiments of the invention relate to an alternativeendgame strategy using a CMV-based polio vaccine that has all thepositive attributes of OPV, but with no possibility for reversion sinceit is not based on a poliovirus genetic background.

OPV was chosen for global poliovirus eradication due to its suitabilityfor use in all nations, both rich and poor. CMV potentially shares allthe characteristics that made OPV the vaccine of choice for globalpoliovirus eradication, but without the down-side of OPV reversion: 1)the capacity for oral administration by medically untrained individuals,2) inexpensive manufacturing without costly chemical inactivation stepsand quality control associated with IPV, and 3) high immunogenicity,inducing mucosal immunity (cellular and antibody) that can blockpoliovirus replication in the gut. CMV enjoys several additionalbeneficial qualities for use as a polio vaccine. CMV can re-infecthealthy individuals regardless of CMV seropositivity.

This ability to re-infect regardless of pre-existing vaccine vectorimmunity is a decided advantage over other vaccines including OPV, whereimmunity against the vector or cross-reactivity with pathogens presentin the environment (i.e., enteroviruses for OPV), severely limits the‘take’ of the vaccine. CMV is also benign in healthy individuals, andimmunogenicity does not require CMV replication. However, CMV does causedisease in immunosuppressed individuals, such as transplant patients andneonates. In these immunosuppressed populations, CMV replication is anabsolute requirement for pathogenicity: no replication equals nodisease. The dissociation of CMV immunity from CMV replication is acritical finding as it suggests that a replication-defective CMV-basedpolio vaccine will be immunogenic and safe in all human populationsregardless of immune-status. Finally, CMV vaccines work. Recent studiesin rhesus macaque (RMs) show that rhesus CMV (RhCMV) expressing simianimmunodeficiency (SIV) antigens induce protection against SIV infection.This is the first vaccine against SIV or HIV that has been shown toinduce protection against infection. Moreover, protection against SIVwas long-lived (observed at >486 days post-vaccination).

Embodiments of the invention relate to assessing the ability of aMCMV-based polio vaccine expressing a protective target antigen frompoliovirus type 1 to induce protective immunity against poliovirus inmice. A recombinant MCMV stably expressing full-length viral protein 1(VP1) from poliovirus type 1 (MCMV/VP1PV1) is constructed (FIG. 27).Following immunological characterization of MCMV/VP1PV1, protectiveefficacy of MCMV/VP1PV1 against poliovirus type 1 in the mousepoliovirus receptor (PVR) transgenic (Tg) mouse challenge model will bedetermined. A MCMV-based poliovaccine is only used as a‘proof-of-concept’ for development of a human CMV-based poliovirusvaccine for use in humans.

FIG. 27 shows that VP1 in MCMV/VP1_(PV1) is epitope tagged (V5) for easeof detection. MCMV/VP1_(PV1) was serial passaged multiple times inmurine embryo fibroblasts (MEFs) Cells are harvested at the time ofmaximum cytopathic effect (CPE) for analysis of VP1 expression based onanti-V5 reactivity. Serial passage numbers are indicated above the imageof the gel.

Other embodiments of the invention relate to assessing the ability of adissemination-defective version of MCMV/VP1PV1 to induce protectiveimmunity against poliovirus type 1 in the PVR Tg model. Adissemination-defective version of MCMV/VP1PV1 (for example,MCMVΔgL/VP1PV1) will be made by deletion of an essential MCMV gene(glycoprotein L, gL) by standard bacterial artificial chromosome (BAC)recombination. This strategy, followed by complementation on agL-expressing cell line, has been shown to be a straightforwardtechnique for making dissemination-defective CMV viruses (Snyder, C. M.,J. E. Allan, E. L. Bonnett, C. M. Doom, and A. B. Hill. 2010.Cross-presentation of a spread-defective MCMV is sufficient to prime themajority of virus-specific CD8+ T cells. PLoS One 5:e9681.) (Bowman, J.J., J. C. Lacayo, P. Burbelo, E. R. Fischer, and J. I. Cohen. 2011.Rhesus and human cytomegalovirus glycoprotein L are required forinfection and cell-to-cell spread of virus but cannot complement eachother. J Virol 85:2089-99). Immunogenicity and protective efficacy ofMCMVΔgL/VP1PV1 against poliovirus type 1 challenge are determined asdescribed above.

CMV vectors may express a variety of different target antigenssuggesting that CMV/VP1PV1 vectors will induce substantial and highlydurable VP1-specific CD4 and CD8 T cell responses. The ability of OPV,but not IPV, to prevent establishment of poliovirus infection in the gutunderlines the importance of cellular immunity for poliovirus control.CMV/VP1PV1 vectors may induce durable levels of VP1-specific antibodiesand these vectors are further tested to determine if these antibodiesare neutralizing. Both protein and synthetic peptides from VP1 are ableto induce biologically significant levels of neutralizing antibodies(albeit at lower levels than induced by intact virus). The antibodyresponse induced by CMV/VP1PV1 vectors may be neutralizing, but perhapsto a lower level than observed with OPV or IPV vaccination. SinceCMV/VP1PV1 vectors are expected to induce high levels of T cells(systemic as well as mucosal) and a biologically significant level ofVP1-specific neutralizing antibodies, both CMV/VP1PV1 and CMVΔgL/VP1PV1may protect against poliovirus type 1 challenge.

Embodiments of the invention relate to establishing that a safe,dissemination-defective CMV-based vector can induce protective immunityagainst poliovirus type 1, determine whether replicating CMV-basedvector (MCMV/VP1PV1) is immunogenic and efficacious against poliovirustype 1 challenge and using MCMVΔgL/VP1PV1 to establish thatdissemination is not required for immunogenicity or efficacy of aCMV-based polio vaccine. Further embodiments relate to demonstratingthat a dissemination-defective CMV-based vector induces protectiveimmunity against poliovirus type 1 hence establishing that CMVdissemination is not required for vaccine efficacy. This finding ‘pavesthe way’ for development of a safe CMV-based polio vaccine for use inall potential human target populations worldwide. Non-human primatepoliovirus models were crucial during OPV and IPV development, and willbe critical for moving CMV-based polio vaccine towards human trials.Additional embodiments also relate to determining immunogenicity andprotective efficacy of dissemination-defective RhCMVΔgL/VP1PV1 in RM anddetermining whether monovalent RhCMVΔgL/VP1 vectors each expressing aVP1 gene from one of the three poliovirus serotypes, or a singlemultivalent RhCMVΔgL/VP1 expressing all 3 serotypes together can induceprotection against all 3 poliovirus serotypes. The construction of anhuman HCMVΔgL/VP1 version of most efficacious trivalent vaccine forhuman Phase I trials is also encompassed in this invention.

Example 7

A Cytomegalovirus-based Vaccine Encoding a Single Ebola VirusNucleoprotein CTL Epitope Confers Protection Against Ebola Virus. ThisExample demonstrates the ability of a CMV-based vaccine approach toprotect against an highly virulent human pathogen.

Summary: In the present Example Applicants have constructed a MCMV-basedEBOV vector expressing a single CTL epitope from NP of Zaire ebolavirusZEBOV (MCMV/ZEBOV-NP_(CTL)). MCMV/ZEBOV-NP_(CTL) was shown to be highlyimmunogenic, inducing durable, multi-functional CD8⁺ CTL responsesagainst ZEBOV NP in multiple strains of mice. Importantly,MCMV/ZEBOV-NP_(CTL) conferred protection against lethal ZEBOV challengeto a comparable level as a standard ‘benchmark’ EBOV vaccine. Absence ofneutralizing antibodies in protected animals identified protection asbeing T cell-mediated.

Background: Human outbreaks of hemorrhagic disease caused by Ebola virus(EBOV) are a serious human health concern. EBOV, a member of theFiloviridae family, causes rapidly progressing viral hemorrhagic feverculminating in multi-organ failure, shock and death [Feldmann H,Geisbert T W (2010) Ebola haemorrhagic fever. Lancet]. EBOV can besubdivided into four distinct and a fifth putative species [Feldmann H,Geisbert T W, Jahrling P B, al. e (2004) In: Fauquet C, Mayo M A,Maniloff J, Desselberger U, Ball L A, editors. Virus Taxonomy: VIIIthReport of the International Committee on Taxonomy of Viruses. London:Elsevier/Academic Press. pp. 645-653, Towner J S, Sealy T K, Khristova ML, Albarino C G, Conlan S, et al. (2008) Newly discovered ebola virusassociated with hemorrhagic fever outbreak in Uganda. PLoS Pathog 4:e1000212]. EBOV species differ in level of virulence, with Zaireebolavirus (ZEBOV) being the most virulent (80-90% mortality) [SanchezA, Geisbert T W, Feldmann H (2006) Filoviridae: Marburg and EbolaViruses. In: Knipe D M, Howley P M, editors. Fields Virology. 5th ed.Philadelphia: Lippincott Williams & Wilkins. pp. 1409-1448]. Theincreasing frequency of outbreaks in endemic areas of Africa, combinedwith potential for accidental and deliberate introduction intonon-endemic nations makes EBOV an ever-increasing global health concern.Potential for rapid dissemination to non-endemic countries wasdemonstrated in 2008 by importation of Marburg virus (a filovirusclosely related to EBOV) to the US [WHO (2009) Case of MarburgHaemorrhagic Fever imported into the United States] and Netherlands [WHO(2008) Case of Marburg Haemorrhagic Fever imported into the Netherlandsfrom Uganda] by tourists infected in Uganda.

A number of candidate EBOV vaccines have been developed that areprotective against infection in animal models [Falzarano D, Geisbert TW, Feldmann H (2011) Progress in filovirus vaccine development:evaluating the potential for clinical use. Expert review of vaccines 10:63-77, Geisbert T W, Bausch D G, Feldmann H (2010) Prospects forimmunisation against Marburg and Ebola viruses. Reviews in medicalvirology 20: 344-357]. Replication-defective adenovirus expressing EBOVglycoprotein (GP) alone [Sullivan N J, Geisbert T W, Geisbert J B,Shedlock D J, Xu L, et al. (2006) Immune protection of nonhuman primatesagainst Ebola virus with single low-dose adenovirus vectors encodingmodified GPs. PLoS Med 3: e177] or in combination with nucleoprotein(NP) [Sullivan N J, Geisbert T W, Geisbert J B, Xu L, Yang Z Y, et al.(2003) Accelerated vaccination for Ebola virus haemorrhagic fever innon-human primates. Nature 424: 681-684], virus-like particles comprisedof virus matrix protein (VP40) and GP with or without NP [Warfield K L,Swenson D L, Olinger G G, Kalina W V, Aman M J, et al. (2007) Ebolavirus-like particle-based vaccine protects nonhuman primates againstlethal Ebola virus challenge. The Journal of infectious diseases 196Suppl 2: S430-437, Swenson D L, Warfield K L, Negley D L, Schmaljohn A,Aman M J, et al. (2005) Virus-like particles exhibit potential as apan-Filovirus vaccine for both Ebola and Marburg viral infections.Vaccine 23: 3033-3042], and replication-competent vesicular stomatitisvirus (VSV) expressing GP [Jones S M, Feldmann H, Stroher U, Geisbert JB, Fernando L, et al. (2005) Live attenuated recombinant vaccineprotects nonhuman primates against Ebola and Marburg viruses. Nat Med11: 786-790, Geisbert T W, Geisbert J B, Leung A, Daddario-DiCaprio K M,Hensley L E, et al. (2009) Single-injection vaccine protects nonhumanprimates against infection with marburg virus and three species of ebolavirus. J Virol 83: 7296-7304] are all able to consistently induceprotective immunity in small animal and non-human primate (NHP) models.Oral immunization with the VSV-based vaccine has been shown to induceprotection in mice [Jones S M, Stroher U, Fernando L, Qiu X, Alimonti J,et al. (2007) Assessment of a vesicular stomatitis virus-based vaccineby use of the mouse model of Ebola virus hemorrhagic fever. J Infect Dis196 Suppl 2: S404-412], leading to the suggestion of its use for foodbaiting [Groseth A, Feldmann H, Strong J E (2007) The ecology of Ebolavirus. Trends Microbiol 15: 408-416, Dolgin E (2008) Baiting Ebola. TheScientist 22: 22].

A cytomegalovirus (CMV)-based vaccine offers an alternative approach.CMV is one of the most immunogenic viruses known [Sylwester A W,Mitchell B L, Edgar J B, Taormina C, Pelte C, et al. (2005) Broadlytargeted human cytomegalovirus-specific CD4+ and CD8+ T cells dominatethe memory compartments of exposed subjects. J Exp Med 202: 673-685],inducing a characteristic immune response that is highly enriched for‘effector’ (T_(EM)) T cells [Hansen S G, Vieville C, Whizin N,Coyne-Johnson L, Siess D C, et al. (2009) Effector memory T cellresponses are associated with protection of rhesus monkeys from mucosalsimian immunodeficiency virus challenge. Nature medicine 15: 293-299].These cells localize predominantly to extra-lymphoid mucosal sites, andare functionally primed for immediate anti-pathogen effector function[Kaech S M, Wherry E J (2007) Heterogeneity and cell-fate decisions ineffector and memory CD8+ T cell differentiation during viral infection.Immunity 27: 393-405]. A CMV-based vaccine may be related to preventionof systemic infection of rhesus macaques by simian immunodeficiencyvirus (SIV), a NHP model for HIV, which is the first vaccine to preventacquisition of fully pathogenic SIV [Hansen S G, Vieville C, Whizin N,Coyne-Johnson L, Siess D C, et al. (2009) Effector memory T cellresponses are associated with protection of rhesus monkeys from mucosalsimian immunodeficiency virus challenge. Nature medicine 15: 293-299].

To assess the potential of CMV for development as a vaccine againstEBOV, Applicants designed a murine cytomegalovirus (MCMV)-based EBOVvaccine (MCMV/ZEBOV-NP_(CTL)) expressing a CD8⁺ CTL epitope from ZEBOVNP (43-VYQVNNLEEIC-53 (SEQ ID NO: 15); NP₄₃) [Wilson J A, Hart M K(2001) Protection from Ebola virus mediated by cytotoxic T lymphocytesspecific for the viral nucleoprotein. J Virol 75: 2660-2664, Olinger GG, Bailey M A, Dye J M, Bakken R, Kuehne A, et al. (2005) Protectivecytotoxic T-cell responses induced by venezuelan equine encephalitisvirus replicons expressing Ebola virus proteins. J Virol 79:14189-14196, Simmons G, Lee A, Rennekamp A J, Fan X, Bates P, et al.(2004) Identification of murine T-cell epitopes in Ebola virusnucleoprotein. Virology 318: 224-230] fused to a non-essential MCMVprotein, IE2 (FIG. 1 a). MCMV/ZEBOV-NP_(CTL) was constructed by E/Trecombination using a bacterial artificial chromosome (BAC) containingthe MCMV genome (pSM3fr) [Hansen S G, Vieville C, Whizin N,Coyne-Johnson L, Siess D C, et al. (2009) Effector memory T cellresponses are associated with protection of rhesus monkeys from mucosalsimian immunodeficiency virus challenge. Nature medicine 15: 293-299,Wagner M, Jonjic S, Koszinowski U H, Messerle M (1999) Systematicexcision of vector sequences from the BAC-cloned herpesvirus genomeduring virus reconstitution. J Virol 73: 7056-7060]. IndependentpMCMV/ZEBOV-NP_(CTL) clones (5A1 and 5D1) were selected forcharacterization. Restriction enzyme digestion followed byelectrophoresis showed no gross genomic rearrangements compared towild-type (WT) parental BAC (FIG. 32). Viruses were reconstituted bytransfection of BAC DNA into murine embryo fibroblasts (MEFs). In vitrogrowth analysis of reconstituted viruses showed replication kineticscomparable to WT MCMV (FIG. 33).

To assess the level of NP-specific CD8⁺ CTL responses induced byMCMV/ZEBOV-NP_(CTL), Applicants performed immunogenicity studies inH2^(b)-restricted 129S1/SvlmJ/Cr mice. Mice (n=5/group) were immunizedintraperitoneally (i.p.) with MCMV/ZEBOV-NP_(CTL) (clone 5A1 or 5D1),MCMV/PSA (clone 3-1) (a control MCMV expressing an irrelevantH2^(b)-restricted epitope from human prostate-specific antigen, PSA[Pavlenko M, Leder C, Roos A K, Levitsky V, Pisa P (2005) Identificationof an immunodominant H-2D(b)-restricted CTL epitope of human PSA.Prostate 64: 50-59]), WT MCMV or diluent (Mock). Following a ‘boost’using an identical inoculum at week 4, splenocytes were harvested atweek 8 for analysis of T cell responses (FIG. 28B). Antigen-specific Tcells were analyzed by intracellular cytokine staining (ICS) after a 6hour in vitro incubation with peptides representing differentH2^(b)-restricted epitopes. All MCMV/ZEBOV-NP_(CTL) vaccinated miceexhibited significant CD8⁺ CTL responses against ZEBOV NP (FIG. 28B).The level of NP responses elicited by 5A1 and 5D1 were not significantlydifferent, and were considered together as a single data set. The ZEBOVNP-specific T cell responses induced were substantial (mean=2.83% oftotal CD8⁺ T cells; range=0.32 to 5.99%), CD8⁺ (no response in CD4⁺ cellcompartment), and specific (directed against ZEBOV NP, but not PSAcontrol). CD8⁺ CTLs induced against ZEBOV NP were polyfunctional,expressing both IFNγ and TNFα (FIG. 28C). All mice exceptmock-vaccinated controls had CD8⁺ CTLs directed against the MCMV-encodedM45 protein.

A unique characteristic of CMV-induced immune responses is their‘inflation’ over time with maturation into stable ‘effector’ T cell(T_(EM)) memory that persists for life [Klenerman P, Dunbar P R (2008)CMV and the art of memory maintenance. Immunity 29: 520-522]. Comparedto classical central T cell memory (T_(CM)) induced by acute ornon-replicating vectors such as vaccinia virus and adenovirus, T_(EM)are localized to mucosal epithelial effector sites, and have immediateeffector function [Cheroutre H, Madakamutil L (2005) Mucosal effectormemory T cells: the other side of the coin. Cell Mol Life Sci 62:2853-2866, Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A (1999)Two subsets of memory T lymphocytes with distinct homing potentials andeffector functions. Nature 401: 708-712]. To determine the durability ofZEBOV NP-specific T cell responses from a single MCMV/ZEBOV-NP_(CTL)inoculation, mice (n=14) were vaccinated (i.p.) withMCMV/ZEBOV-NP_(CTL), and peripheral T cell responses were followedlongitudinally. NP-specific CD8⁺ T cell responses gradually accumulatedto high levels and persisted (increasing from 0.79% at week 8, to 3.08%at 33 weeks following the single inoculation) (FIG. 29). Althoughdelayed, the NP-specific CTL response was comparable in kinetics ofinduction and magnitude to the T_(EM)-biased ‘inflationary’ responsedirected against MCMV M38 [Munks M W, Cho K S, Pinto A K, Sierro S,Klenerman P, et al. (2006) Four distinct patterns of memory CD8 T cellresponses to chronic murine cytomegalovirus infection. J Immunol 177:450-458, Karrer U, Sierro S, Wagner M, Oxenius A, Hengel H, et al.(2003) Memory inflation: continuous accumulation of antiviral CD8+ Tcells over time. J Immunol 170: 2022-2029]. Importantly, these resultsshow that a CMV-based EBOV vaccine can induce high levels of CD8⁺ Tcells against an EBOV antigen that increase with time and are durable.

To determine whether MCMV/ZEBOV-NP_(CTL) was able to induce protectiveimmunity against lethal ZEBOV challenge, Applicants performed challengestudies in C57BL/6 mice using mouse-adapted ZEBOV (ma-ZEBOV) as thechallenge virus [Jones S M, Stroher U, Fernando L, Qiu X, Alimonti J, etal. (2007) Assessment of a vesicular stomatitis virus-based vaccine byuse of the mouse model of Ebola virus hemorrhagic fever. J Infect Dis196 Suppl 2: S404-412, Bray M, Davis K, Geisbert T, Schmaljohn C,Huggins J (1998) A mouse model for evaluation of prophylaxis and therapyof Ebola hemorrhagic fever. J Infect Dis 178: 651-661]. The ma-ZEBOV isuniformly lethal in unvaccinated mice, which succumb 5-7 dayspost-challenge [Jones S M, Stroher U, Fernando L, Qiu X, Alimonti J, etal. (2007) Assessment of a vesicular stomatitis virus-based vaccine byuse of the mouse model of Ebola virus hemorrhagic fever. J Infect Dis196 Suppl 2: S404-412, Bray M, Davis K, Geisbert T, Schmaljohn C,Huggins J (1998) A mouse model for evaluation of prophylaxis and therapyof Ebola hemorrhagic fever. J Infect Dis 178: 651-661]. Four groups ofmice (n=20/group) were immunized with MCMV/ZEBOV-NP_(CTL) 5A1 or 5D1,MCMV WT or diluent, and boosted at week 4 (FIG. 30). At week 8,splenocytes from 6 mice/group were analysed for T cell responses. 5A1and 5D1 induced comparable responses against NP, enabling mice receivingeither clone to be considered as a single data set. MCMV/ZEBOV-NP_(CTL)induced considerable levels of CD8⁺ T cells against ZEBOV NP (mean=1.34%of total CD8⁺ T cells; range=0.05 to 2.68%). These results also showthat the ability of MCMV/ZEBOV-NP_(CTL) to induce NP-specific T cells isindependent of the mouse strain (FIG. 28C and FIG. 30). All mice exceptmock-vaccinated controls had CD8⁺ CTLs directed against MCMV-encoded M38and M45.

At week 10, the remaining mice (n=14/group) were challenged i.p. with10³ LD₅₀ of ma-ZEBOV. An additional group (n=14) that had receivedVSVΔG/ZEBOVGP (5×10⁵ pfu; i.p.), which confers high levels of protectionagainst ma-ZEBOV, was included as a control for vaccine protection[Jones S M, Stroher U, Fernando L, Qiu X, Alimonti J, et al. (2007)Assessment of a vesicular stomatitis virus-based vaccine by use of themouse model of Ebola virus hemorrhagic fever. J Infect Dis 196 Suppl 2:S404-412.]. ZEBOV disease was then monitored on the basis of survival,morbidity based on clinical signs (ruffled fur, hunched posture,paralysis and weight loss) and viremia. Mock and MCMV WT vaccinatedcontrols exhibited normal ZEBOV disease, with significant morbidity(FIG. 31B); they succumbed to infection within the normal time (mode=7days) post-challenge (FIG. 31A). In contrast, MCMV/ZEBOV-NP_(CTL)vaccinated mice showed no evidence of ZEBOV disease, with 100% survivaland no signs of morbidity (FIGS. 31A-B). As a quantitative analysis ofvaccine efficacy, viremia at day 4 post-challenge (peak of ZEBOV viremiain mice) was measured in a subset of mice (n=3-4/group) harvested atthis time (FIG. 31C). MCMV/ZEBOV-NP_(CTL) vaccination resulted in aprofound level of control, with viremia reduced to levels observed forthe VSVΔG/ZEBOVGP controls. Specifically, 5 of 8 mice showed a completecontrol of ZEBOV viremia down to undetectable levels (sterilizingimmunity). The remaining 3 mice showed a 2.5-log reduction in viremiacompared to WT MCMV vaccinated controls. MCMV/ZEBOV-NP_(CTL) vaccinationdoes not induce ZEBOV neutralizing antibodies, as would be expected fromexpression of a single 11-mer CTL epitope of NP. However, low levels ofma-ZEBOV replication following challenge could feasibly have inducedneutralizing antibodies which would positively impact diseaseresolution. To determine whether protection resulted entirely from Tcell-mediated control, Applicants measured ma-ZEBOV neutralizingactivity in sera from a randomly selected subset (n=6) of protectedMCMV/ZEBOV-NP_(CTL) mice at 28 days post-challenge. VSVΔG/ZEBOVGPcontrol mice had low, but detectable levels of neutralizing activityfollowing challenge [Jones S M, Stroher U, Fernando L, Qiu X, AlimontiJ, et al. (2007) Assessment of a vesicular stomatitis virus-basedvaccine by use of the mouse model of Ebola virus hemorrhagic fever. JInfect Dis 196 Suppl 2: S404-412]. In contrast, neutralizing activitywas not detected in any convalescent serum from MCMV/ZEBOV-NP_(CTL)vaccinated mice demonstrating that protection was mediated solely byCD8⁺ T cells (FIG. 33).

Conclusions: Applicants demonstrate that a CMV-based vaccine can affordprotection against ZEBOV—the first study to show that a CMV-basedvaccine can protect against any human disease. The level of protectionwas profound, with ZEBOV control comparable to that achieved by one ofthe ‘benchmark’ vaccines, VSVΔG/ZEBOVGP. This level of ZEBOV control,achieved using a CMV vector expressing only a single CTL epitope,underscores the potential of this vaccine platform.

FIG. 28 depicts T cell responses following immunization withMCMV/ZEBOV-NP_(CTL). (a) Schematic representation ofMCMV/ZEBOV-NP_(CTL). An H2^(b)-restricted T cell epitope from ZEBOV NP(VYQVNNLEEIC (SEQ ID NO: 15)) was fused ‘in-frame’ to the carboxylterminus of MCMV IE2 (M122) generating the recombinant MCMV,MCMV/ZEBOV-NP_(CTL). MCMV IE2 is a non-essential protein. (b)129S1/SvlmJ/Cr (H2^(b)-restricted) mice (n=5/group) were immunizedintraperitoneally (i.p.) using 5×10⁵ pfu of the following: one of twoindependent clones of MCMV/ZEBOV-NP_(CTL) (5A1 and 5D1), MCMV/PSA (clone3-1) (a comparable MCMV vector expressing IE2 fused to anH2^(b)-restricted epitope from human prostate specific antigen, PSA), WTMCMV, or diluent (mock control). Mice were boosted at week 4, andsplenocytes were harvested for analysis of T cell responses at week 8. Tcells were analysed by using intracellular cytokine staining (ICS) witha 6 hour incubation in the presence of brefeldin A (BFA) with peptide(or anti-CD3 MAb, for total T cell response). Levels of responding (IFNγand TNFα double-positives) CD8⁺ (top) and CD4⁺ (bottom) cells inindividual mice are shown. All MCMV/ZEBOV-NP_(CTL) immunized mice (n=10)showed significant CD8-restricted T cell responses against the NP targetantigen. (c) Typical responses from MCMV/ZEBOV-NP_(CTL) vaccinated mice.The majority of ZEBOV NP-responding T cells are polyfunctional(expressing both IFNγ and TNFα) and are specific for the NP epitope (notobserved following incubation with the PSA peptide or unstimulatedcontrols). Consistent with MCMV infection, all mice demonstrate T cellresponses to MCMV M45. T cell responses directed against M45 are knownto be ‘non-inflationary’, generally representing <1% of total CD8⁺ Tcells during chronic MCMV infection. Error bars show the standarddeviation (s.d.).

FIG. 29 depicts Kinetic analysis of CD8⁺ T cell response toMCMV/ZEBOV-NP_(CTL). 129S1/SvlmJ/Cr H2^(b)-restricted mice (n=14) wereimmunized (i.p.) with a single dose (1×10⁵ pfu) of MCMV/ZEBOV-NP_(CTL)(clone 5D1). At times indicated, mice were bled and peripheral T cellresponses were measured in pooled blood by using ICS with a 6 hourincubation in the presence of BFA with peptides. All responses werenormalized against cells stimulated in the absence of peptide. Responsesare against ZEBOV NP (black), or MCMV M38 (grey) and M45 (white). Errorbars show the s.d.

FIG. 30 depicts MCMV/ZEBOV-NP_(CTL) induces a ZEBOV-specific T cellresponse in C57BL/6 mice. C57BL/6 H2^(b)-restricted mice (n=6/group)were vaccinated (i.p.) using 5×10⁵ pfu of MCMV/ZEBOV-NP_(CTL) clone 5A1or 5D1. Control groups received either MCMV WT, or diluent (Mock). Atweek 4, mice were boosted as before. At week 8 mice were harvested foranalysis of splenocyte T cell responses by ICS using a 6 hour incubationin the presence of BFA with indicated peptide. MCMV-specific CD8⁺ T-cellresponses against MCMV M45 and M38 were used as markers of MCMVinfection. PSA peptide served as an H2^(b)-restricted epitopespecificity control. Responding CD8⁺ cells shown are IFNγ and TNFαdouble-positives. Mice groups presented in this figure were vaccinatedin parallel with mice groups (n=14/group) used to ascertain protectiveefficacy of vaccination regimen shown in FIG. 4. Error bars show thes.d.

FIG. 31 depicts Protective efficacy of MCMV/ZEBOV-NP_(CTL). Groups ofC57BL/6 mice (n=14) were vaccinated by i.p. administration of 5×10⁵ pfuof either MCMV/ZEBOV-NP_(CTL) (clones 5A1 or 5D1), MCMV WT, or diluentDMEM (Mock), followed by an identical boost at week 4. An additionalgroup received VSVΔG/ZEBOVGP as a positive control for vaccine efficacy.At week 10, mice were challenged with 10³ LD₅₀ ma-ZEBOV (i.p.). Datarepresent (a) Percent survival. (b) Body weight change over timepost-challenge (error bars show the s.d.). (c) Viremia levels in 3-4mice harvested at time of peak viremia (day 4) (mean viremia levels foreach group are shown in parentheses). For body weight, groups wereweighed daily until 14 days post-EBOV challenge, or until all animals ina group had succumb to ZEBOV disease. MCMV/ZEBOV-NP_(CTL) vaccinationhad a significant impact on survival from ma-ZEBOV challenge compared toMCMV WT controls (p<0.0001) using a log-rank test. Analysis of ma-ZEBOVviremia show a comparable level of viremia control betweenMCMV/ZEBOV-NP_(CTL) vaccinated groups, compared to MCMV WT controls(p<0.0001). No significant differences were observed betweenVSVΔG/ZEBOVGP and MCMV/ZEBOV-NP_(CTL) vaccinated groups in survival,morbidity (weight loss), or viremia (p=0.3).

FIG. 32 depicts genomic characterization of MCMV/ZEBOV-NP_(CTL). BAC DNAfrom two independent clones of MCMV/ZEBOV-NP_(CTL) (5A1 and 5D1) weredigested with EcoRI followed by electrophoresis. The comparable digestpattern between MCMV/ZEBOV-NP_(CTL) BAC clones and the MCMV WT BAC showsthe lack of any gross genomic rearrangement.

FIG. 33 depicts multi-step growth analysis of MCMV/ZEBOV-NP_(CTL) (5A1and 5D1). MEFs were infected at a MOI of 0.1 with either 5A1, 5D1 or WTMCMV. Supernatant was collected at days indicated post-infection andtitered by standard plaque assay. The assay was performed in triplicateand standard deviation is shown.

Example 8 Recombinant CMV Vector with a Deletion in the Gene EncodingUL82 (pp71)

This example relates to a replication-impaired RhCMV that was able toinfect CMV-naïve animals and induce a CMV-specific immune response. Arecombinant RhCMV lacking the tegument protein pp71 (encoded by Rh110)was generated. The homologous pp71 protein encoded by UL82 of HCMV waspreviously shown to stimulate viral immediate early gene expression andgrowth of HCMV lacking pp71 is reduced by several orders of magnitude invitro (Cantrell et al., J Virol 80:6188-6191, 2006; Preston and Nicholl,J Gen Virol 87:1113-1121, 2006; Saffert and Kalejta, J Virol80:3863-3871). Similarly, RhCMV lacking pp71 (RhCMVΔpp71, RhCMVΔRh110)grows only poorly on rhesus fibroblasts, but normal titers were restoredin rhesus fibroblasts stably expressing pp71 from a retroviral vector.FIG. 36 shows the deletion of pp71 impairs viral release from normalfibroblasts, but not from fibroblasts expressing pp71. Two differentvectors are generated: RhCMVΔRh110 has a gene deletion of Rh110 whichencodes for pp71, in RhCMVΔRh110 retanef the Rh110 gene is replaced withan expression cassette for a fusion protein of the proteins rev tat andnef from simian-immunodeficiency virus (SIV). RhCMV-WT refers to theparental (wiltype) virus. In the top panel rhesus fibroblasts that arelife-extended by telomerase expression (tRFs) are infected at day 0 with0.01 viruses per cell (multiplicity of infection (MOI)=0.01). The cellsupernatant is collected on each of the indicated days and the viraltiter is determined on tRFs that stably express pp71. The data indicatesthat pp71-deletion reduced titers by more than 1000 fold consistent witha severe deficiency in viral production. To confirm that this growthdefect is due to lack of the pp71 protein, pp71-expressing tRFs areinfected at an MOI of 0.01 and the supernatant is collected. As shown inthe lower panel, the same amount of virus was released when thesecomplementing cells are infected with ΔRh110 or WT viruses.

Infection of pp71-expressing RF allowed generation of high-titer stockfor infection of CMV-naïve RM. Two sero-negative RM were infected s.c.with 10⁷ PFU of RhCMVΔpp71. The cellular immune response to RhCMV wasmonitored in bronchioalveolar lavages (BAL) and in peripheral bloodmononuclear cells (PBMC) at weekly intervals using intracellularcytokine staining as described (Hansen et al., Science328(5974):102-106, 2010; Hansen et al., Nat Med 15:293-299, 2009).Strikingly, both animals developed a significant immune response to CMVwithin two weeks of infection that is comparable to historic controlanimals infected with RhCMV-WT (FIG. 20).

FIG. 37 (upper panel) further shows that the T cell response remainedstable over 245 days. Two sero-negative rhesus macaques (RM) areinoculated s.c. with 10⁷ plaque forming units (PFU) of RhCMV ΔRh110 atday 0. The CMV-specific CD8+ and CD4+ T cell response against RhCMVlysate is measured by ICCS in PBMC and BAL at the indicated intervals.The immune response is comparable to that of animals infected with RhCMVWT (black lines). These results indicate that despite its severeattenuation, pp71-deleted virus was able to generate a long-lastingWT-like immune response.

FIG. 37 (lower panels) demonstrates that replication-impaired RhCMV isnot secreted from infected animals. To determine whether pp71-deletedRhCMV was secreted from the two CMV-negative animals infected withRhCMVΔRh110 (Δpp71) virus was concentrated from urine samples andco-cultured with pp71-expressing fibroblasts. For control, twoCMV-positive animals were infected with WT-RhCMV expressing RhCMVgag.Expression of SIVgag, RhCMV protein IE or the cellular protein GAPDH(included as loading control) is determined from viral cocultures byimmunoblot using specific antibodies (S. G. Hansen et al. Science 328,5974 (2010)). The two animals infected with RhCMV(gag) secreted RhCMV(as shown by IE expression) because they are CMV-positive at the onsetof the experiment. At day 56, these animals also secrete SIVgagexpressing RhCMV indicating infection. In contrast, the two CMV-negativeRM infected with ΔRh110 did not secrete RhCMV as indicated by theabsence of IE-positive cocultures up to the last time point tested sofar (day 231). This indicates that ΔRh110 is attenuated in vivo yetretains the same immunogenicity over the entire time of the experiment(245 days). This result further indicates that the pp71-deleted virus isunlikely to be transmitted from one animal to another. Thus,pp71-deleted vectors are replication-impaired and spread-deficient.

This example further relates to a replication-impaired RhCMV expressinga heterologous antigen that was able to super-infect CMV-positiveanimals and induce a immune response specific to the heterologousantigen. To demonstrate whether pp71-deleted vectors are able tosuper-infect CMV-positive RM and induce a longterm effector memory Tcell response to a heterologous antigen, two essential features ofCMV-vectored vaccines, four RM are inoculated with pp71-deleted vectorsexpressing the SIV antigens rev/tat/nef together with a WT vectorexpressing the SIV-antigen pol. This co-inoculation allows thedetermination of whether there are differences in the kinetics andduration of the T cell response to the SIV antigens expressed by WTversus replication-impaired vectors.

FIG. 38 shows that pp71-deleted CMV vectors expressing a heterologousantigen are able to super-infect CMV-infected animals and induce along-lasting immune response to the heterologous antigen but are notsecreted. Upper Panel: Mean frequencies (+/−SEM) of SIVrev/tat/nef- andSIVpol-specific CD8+ T cells from blood and broncho-alveolar lavage(BAL) lymphocytes of 4 CMV-positive RM inoculated simultaneously withRhCMV lacking pp71 and expressing SIVretanef (Δpp71 RhCMV/rtn, in red)and wildtype vectors expressing SIVpol (wt RhCMV/pol, in blue).SIVrev/nef/tat-specific responses of 6 RM given wt RhCMV/retanef areshown as an additional control for comparison (in black). Responsefrequencies were determined by intra-cellular TNFα and/or IFN-γexpression after stimulation with overlapping rev/tat/nef or polpeptides. SIVpol or SIVretanef responding CD8+ T cells from blood at day133 post-inoculation were also analyzed for memory phenotype—thefraction of the total SIV retanef- or pol-specific response with aphenotype of central memory T cells, T_(CM) (CD28+/CCR7+), transitionalT cells, T_(Trans.EM) (CD28+/CCR7−) and effector memory T cells, T_(EM)(CD28−/CCR7−) are shown. These data clearly show that pp71-deletedvectors retain the ability to super-infect animals already infected CMVand induce a longterm effector memory T cell response to a heterologousantigen.

Lower Panel: Urine co-culture after inoculation of RhCMV-positive RMwith Δpp71 RhCMV/retanef (RM 3/RM4) vs. wt RhCMV/retanef (Con) vectors,respectively. Urine is collected at the designated time points, andRhCMV IE (primary infection) or V5-tagged SIVrev/tat/nef(super-infection) expression is detected by immunoblot of co-culturelysates. These data show that the pp71-deleted vector is not secretedfrom infected animals consistent with spread-deficiency. Taken together,these data demonstrates that immunogenicity of CMV-vectors is notcompromised even when vectors are severely impaired in their ability toreplicate in vivo and in vitro.

Example 9

Applicants observed that multiple genes of CMV can be eliminated withoutcompromising features that Applicants consider essential for vectorefficacy, specifically the ability to super-infect and establishlong-term antigen expression and thus induction of durable effectormemory T cells (T_(EM)) responses. Applicants' data revealed anastoundingly large number of genes that can be deleted withoutcompromising the ability of RhCMV to super-infect and to inducelong-term T_(EM) responses. Specifically, Applicants have constructed apanel of RhCMV/gag recombinants containing large gene deletions (up to10 kb each) that together comprise ˜30 kb (24 genes) (Table 6, FIG. 34).

TABLE 6 RhCMV gene regions non- RhCMV candidates for replacementHomologous HCMV Candidates essential for growth in vivo (% identity toHCMV) (kinetic class) Rh13-Rh29 Rh13.1 (40%), Rh20 (26%), RL11 (L), UL6(?), UL7 (L), UL9 (L), (RL11 family) Rh19 (34%), Rh24 (35%), Rh23 (36%)UL11 (E) Rh111-Rh112 (pp65) Rh112 (35%) UL83 (L) (pp65) Rh191-Rh202Rh190 (33%), Rh192 (24%), Rh196 (29%), US12 (E), US13 (E), US14 (E),US17 (US12 family) Rh198 (36%), Rh199 (32%), Rh200 (29%), (E), US18 (E),US19 (E), US20 (E), Rh201 (40%), Rh202 (58%) US21 (?) Rh214-Rh220 (US28family) Rh220 (37%) US28 (E)

Most of these genes and gene families are conserved in HCMV and areknown not to be essential for HCMV growth in vitro (Yu, D., M. C. Silva,and T. Shenk. 2003. Functional map of human cytomegalovirus AD169defined by global mutational analysis. Proc Natl Acad Sci USA100:12396-401). Deletion of these genes in RhCMV did not affect in vitrogrowth characteristics, super-infection efficiency, persistence,shedding or immunogenicity of RhCMV/gag vectors (FIG. 35), and thus anyindividual gene or gene group in these dispensable regions can clearlybe replaced with heterologous antigens.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

Sequence CWU 0 SQTB SEQUENCE LISTING The patent application contains alengthy “Sequence Listing” section. A copy of the “Sequence Listing” isavailable in electronic form from the USPTO web site. An electronic copyof the “Sequence Listing” will also be available from the USPTO uponrequest and payment of the fee set forth in 37 CFR 1.19(b)(3).

What is claimed is:
 1. A recombinant viral vector comprising a nucleicacid sequence encoding a human cytomegalovirus (HCMV) backbone vectorand at least one human immunodeficiency virus (HIV)-derived antigen,wherein the recombinant viral vector: (a) comprises a deletion in geneUL82 that eliminates expression of a functional pp71 protein, (b) isdeficient in host to host spread, (c) infects a HCMV seropositive hostupon administration of said recombinant viral vector, and (d) inducesand maintains a long-term effector memory T cell response to the atleast one HIV-derived antigen in said seropositive host.
 2. Therecombinant viral vector of claim 1, further comprising a deletion in aHCMV gene region non-essential for growth in vivo.
 3. The recombinantviral vector of claim 2, wherein the non-essential HCMV gene region isselected from the group consisting of: the RL11 family, the pp65 family,the US12 family, and the US28 family.
 4. The recombinant viral vector ofclaim 2, wherein the non-essential HCMV gene region is selected from thegroup consisting of: RL11, UL6, UL7, UL9, UL11, UL78, UL83, US12, US13,US14, US17, US18, US19, US20, US21, and UL28.
 5. The recombinant viralvector of claim 1, further comprising at least one deletion in a HCMVgene required for optimal growth in certain cell types.
 6. Therecombinant viral vector of claim 5, wherein the at least one deletionin a HCMV gene required for optimal growth in certain cell typescomprises a deletion in UL64 or US29, or a combination thereof.
 7. Therecombinant viral vector of claim 5, wherein the recombinant viralvector has a deficient tropism for epithelial cells, the central nervoussystem (CNS), macrophages, or a combination thereof.
 8. The recombinantviral vector of claim 1, further comprising a deletion in at least oneimmune modulatory gene selected from the group consisting of: US2, US3,US4, US5, US6, US7, US8, US9, US10, US11, UL118, UL119, UL36, UL37,UL111a, UL146, and UL147.
 9. The recombinant viral vector of claim 1,wherein the HCMV backbone vector comprises a nucleic acid sequence thatis at least 90%, at least 95%, at least 99%, or 100% identical to thenucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO:
 9. 10.The recombinant viral vector of claim 1, wherein the at least oneHIV-derived antigen is selected from the group consisting of: Gag, Pol,Env, Rev, Tat, and Nef, or an epitope or antigenic fragment thereof. 11.A recombinant viral vector comprising a nucleic acid sequence encoding arhesus cytomegalovirus (RhCMV) backbone vector and at least one simianimmunodeficiency virus (SIV)-derived antigen, wherein the recombinantviral vector: (a) comprises a deletion in gene Rh110 that eliminatesexpression of a functional pp71 protein, (b) is deficient in host tohost spread, (c) infects a RhCMV seropositive host upon administrationof said recombinant viral vector, and (d) induces and maintains along-term effector memory T cell response to the at least oneSIV-derived antigen in said seropositive host.
 12. The recombinant viralvector of claim 11, further comprising a deletion in a RhCMV gene regionnon-essential for growth in viva.
 13. The recombinant viral vector ofclaim 12, wherein the non-essential RhCMV gene region is selected fromthe group consisting of: the RL11 family, the pp65 family, the US12family, and the US28 family.
 14. The recombinant viral vector of claim11, further comprising a deletion in at least one immune modulatory geneselected from the group consisting of: Rh158, Rh159, Rh160, Rh161,Rh162, Rh163, Rh164, Rh165, Rh166, Rh182, Rh183, Rh184, Rh185, Rh186,Rh187, Rh188, and Rh189.
 15. The recombinant viral vector of claim 11,wherein the RhCMV backbone vector comprises a nucleic acid sequence thatis at least 90%, at least 95%, at least 99%, or 100% identical to thenucleic acid sequence of SEQ ID NO:
 1. 16. The recombinant viral vectorof claim 11, wherein the at least one SIV-derived antigen is selectedfrom the group consisting of: Gag, Pol, Env, Rev, Tat, and Nef, or anepitope or antigenic fragment thereof.
 17. A recombinant viral vectorcomprising a nucleic acid sequence encoding a HCMV backbone vector andat least one HIV Gag epitope, wherein the recombinant viral vector: (a)comprises a deletion in gene UL82 that eliminates expression of afunctional pp71 protein, (b) is deficient in host to host spread, (c)infects a HCMV seropositive host upon administration of said recombinantviral vector, and (d) induces and maintains a long-term effector memoryT cell response to HIV Gag.
 18. The recombinant viral vector of claim12, wherein the non-essential RhCMV gene region is selected from thegroup consisting of: Rh13-Rh29, Rh111-Rh112, Rh191-Rh202, Rh214-Rh220,Rh13.1, Rh19, Rh20, Rh23, Rh24, Rh112, Rh190, Rh192, Rh196, Rh198,Rh199, Rh200, Rh201, Rh202, and Rh220.
 19. The recombinant viral vectorof claim 11, further comprising at least one deletion in an RhCMV generequired for optimal growth in certain cell types.
 20. The recombinantviral vector of claim 19, wherein the recombinant vector has a deficienttropism for epithelial cells, the central nervous system (CNS),macrophages, or a combination thereof.
 21. The recombinant viral vectorof claim 17, further comprising at least one HIV-derived antigenselected from the group consisting of: Pol, Env, Rev, Tat, and Nef, oran epitope or antigenic fragment thereof.
 22. The recombinant viralvector of claim 17, further comprising a deletion in a HCMV gene regionnon-essential for growth in vivo.
 23. The recombinant viral vector ofclaim 17, further comprising at least one deletion in a HCMV generequired for optimal growth in certain cell types.
 24. The recombinantviral vector of claim 23, wherein the recombinant vector has a deficienttropism for epithelial cells, the central nervous system (CNS),macrophages, or a combination thereof.